Kits and methods for generating 5&#39; capped rna

ABSTRACT

The present invention relates to kits and methods for efficiently generating 5′ capped RNA having a modified cap nucleotide and for use of such modified-nucleotide-capped RNA molecules. In particular, the present invention provides kits and methods for capping RNA using a modified cap nucleotide and a capping enzyme system, such as poxvirus capping enzyme. The present invention finds use for in vitro production of 5′-capped RNA having a modified cap nucleotide and for in vitro or in vivo production of polypeptides by in vitro or in vivo translation of such modified-nucleotide-capped RNA. The invention also provides methods and kits for capturing or isolating uncapped RNA comprising primary RNA transcripts or RNA having a 5′-diphosphate, and methods and kits for using a capping enzyme system and modified cap nucleotides for labeling uncapped RNA comprising primary RNA transcripts or RNA having a 5′-diphosphate with detectable dye or enzyme moieties.

The present invention is a continuation of U.S. patent application Ser.No. 14/471,649, filed Aug. 28, 2014, which is a continuation of U.S.patent application Ser. No. 14/185,384, filed Feb. 20, 2014, which is acontinuation of U.S. patent application Ser. No. 11/787,352, filed Apr.16, 2007, which claims priority U.S. Provisional Patent Application Ser.No. 60/792,220, filed Apr. 14, 2006, each of which are hereinincorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to kits and methods for efficientlygenerating 5′ capped RNA having a modified cap nucleotide and uses ofsuch modified-nucleotide-capped RNA molecules. The invention can be usedto obtain novel compositions of such modified-nucleotide-capped RNAmolecules. In particular, the present invention provides kits andmethods for capping RNA using a modified cap nucleotide and a cappingenzyme system, such as vaccinia virus capping enzyme. The presentinvention finds use for in vitro production of 5′-capped RNA having amodified cap nucleotide and for in vitro or in vivo production ofpolypeptides by in vitro or in vivo translation of suchmodified-nucleotide-capped RNA for a variety of research, therapeutic,and commercial applications. The invention also provides methods andkits for capturing or isolating uncapped RNA comprising primary RNAtranscripts or RNA having a 5′-diphosphate, such as RNA synthesized invitro or obtained from a biological source, including prokaryotic mRNAthat is in a mixture with other prokaryotic and/or eukaryotic nucleicacids. The method for capturing modified-nucleotide-capped RNA alsoprovides methods and kits for obtaining only type-specific orcondition-specific modified-nucleotide-capped RNA by cap-dependentsubtraction of that portion of the captured modified-nucleotide-cappedRNA in cells of one type or condition that is the same as RNA in cellsof another type or condition. The invention further provides methods andkits for using a capping enzyme system and modified cap nucleotides forlabeling uncapped RNA comprising primary RNA transcripts or RNA having a5′-diphosphate with detectable dye or enzyme moieties.

BACKGROUND OF THE INVENTION

Most eukaryotic cellular mRNA transcripts and most eukaryotic viral mRNAtranscripts are blocked or “capped” at their 5′ terminus. In addition tomRNA, some other forms of eukaryotic RNA, such as but not limited to,small nuclear RNA (“snRNA”) and pre-micro RNA (i.e. “pre-miRNA”, theprimary transcripts that are processed to miRNA) are also capped.

A “cap” is a guanine nucleoside that is joined via its 5′-carbon to atriphosphate group that is, in turn, joined to the 5′-carbon of the most5′-nucleotide of the primary mRNA transcript, and in most eukaryotes,the nitrogen at the 7 position of guanine in the cap nucleotide ismethylated. Such a capped transcript can be represented asm⁷G(5′)ppp(5′)N₁(pN)_(x)-OH(3′), or more simply, as m⁷GpppN₁(pN)_(x),where m⁷G represents the 7-methylguanosine cap nucleoside, ppprepresents the triphosphate bridge between the 5′ carbons of the capnucleoside and the first nucleotide of the primary RNA transcript, andN₁(pN)_(x)-OH(3′) represents the primary RNA transcript, of which N₁ isthe most 5′-nucleotide.

The 5′ caps of eukaryotic cellular and viral mRNAs (and some other formsof RNA) play important roles in RNA stability and processing. Forexample, the cap plays a pivotal role in mRNA metabolism, and isrequired to varying degrees for processing and maturation of an RNAtranscript in the nucleus, transport of mRNA from the nucleus to thecytoplasm, mRNA stability, and efficient translation of the mRNA toprotein.

The 5′ cap structure is involved in the initiation of protein synthesisof eukaryotic cellular and eukaryotic viral mRNAs and in mRNA processingand stability in vivo (e.g., see, Cell 9: 645-653, 1976; Furuichi, etal., Nature 266: 235, 1977; Federation of Experimental BiologistsSociety Letter 96: 1-11, 1978; Prog. Nuc. Acid Res. 35: 173-207, 1988).Specific cap binding proteins exist that are components of the machineryrequired for initiation of translation of an mRNA (e.g., see Cell 40:223-24, 1985; Prog. Nuc. Acid Res. 35: 173-207, 1988). The cap of mRNAis recognized by the translational initiation factor eIF4E (Gingras, etal., Ann. Rev. Biochem. 68: 913-963, 1999; Rhoads, R E, J. Biol. Chem.274: 30337-3040, 1999). Thus, RNA prepared (e.g., in vitro) forintroduction into eukaryotic cells (e.g., via microinjection intooocytes or transfection into cells) should be capped.

Also, many viral RNAs are infectious only when capped, and uncapped RNAsintroduced into cells via transfection or microinjection are rapidlydegraded by cellular RNases (e.g., see Krieg, and Melton, Nucleic AcidsRes. 12: 7057, 1984; Drummond, et al. Nucleic Acids Res. 13: 7375,1979).

The 5′ cap structure provides resistance to 5′-exonuclease activity andits absence results in rapid degradation of the mRNA (e.g., see Mol.Biol. Med. 5: 1-14, 1988; Cell 32: 681-694, 1983). Since the primarytranscripts of many eukaryotic cellular genes and eukaryotic viral genesrequire processing to remove intervening sequences (introns) within thecoding regions of these transcripts, the benefit of the cap also extendsto stabilization of such pre-mRNA. This was demonstrated using mutantsof capping enzymes in the budding yeast Saccharomyces cerevisiae. Forexample, it was shown that the presence of a cap on pre-mRNA enhanced invivo splicing of pre-mRNA in yeast, but was not required for splicing,either in vivo or using in vitro yeast splicing systems (Fresco, L D andBuratowski, S, RNA 2: 584-596, 1996; Schwer, B et al., Nucleic AcidsRes. 26: 2050-2057, 1998; Schwer, B and Shuman, S, RNA 2: 574-583,1996). The enhancement of splicing was primarily due to the increasedstability of the pre-mRNA since, in the absence of a cap, the pre-mRNAwas rapidly degraded by 5′ exoribonuclease (Schwer, B, Nucleic AcidsRes. 26: 2050-2057, 1998). Thus, it is also beneficial that transcriptssynthesized for in vitro RNA splicing experiments are capped.

In vitro, capped RNAs have been reported to be translated moreefficiently than uncapped transcripts in a variety of in vitrotranslation systems, such as rabbit reticulocyte lysate or wheat germtranslation systems (e.g., see Paterson and Rosenberg, Nature 279: 692,1979). This effect is also believed to be due in part to protection ofthe RNA from exoribonucleases present in the in vitro translationsystem, as well as other factors. Therefore the importance of the capcan vary with the particular translation system and its method ofpreparation. In any case, the use of capped transcripts can bebeneficial in many cases.

The synthesis of capped RNA transcripts in vitro provides considerablevalue and importance for a variety of functions and applications, suchas for in vitro and in vivo protein synthesis. In addition to beingcapped, most eukaryotic cellular and viral mRNAs have poly(A) tails ontheir 3′ termini. There appears to be a synergy between the 3′ poly(A)tail and the 5′-cap in increasing mRNA stability and translation.Without being bound by theory, this synergy is believed to involve aninteraction between the poly(A) binding protein and the N-terminal partof the eIF4G cap binding protein, leading to mRNA circularization via acomplex between the cap, the cap binding protein, the poly(A) bindingprotein, and the poly(A) tail. Some aspects and applications of thissynergy are presented and discussed by Mockey, M et al. (Biochem.Biophys. Res. Comm. 340: 1062, 2006).

While capped mRNA remains in the cytoplasm after being exported from thenucleus, some other RNAs, such as some snRNAs have caps that are furthermethylated and then imported back into the nucleus, where they areinvolved in splicing of pre-mRNA (Mattaj, Cell 46: 905-911, 1986; Hammet al., Cell 62: 569-577, 1990; Fischer, et al., J. Cell Biol. 113:705-714, 1991). Transcripts with trimethylated caps have been shown tobe translated with higher efficiency using Ascaris lumbicoides extractsin vitro (Maroney et al., RNA 1: 714-723, 1995).

In vivo, capping of a 5′-triphosphorylated primary mRNA transcriptoccurs via several enzymatic steps (e.g., see Martin, S A et al., J.Biol. Chem. 250: 9322, 1975; Myette, J R and Niles, E G, J. Biol. Chem.271: 11936, 1996; M A Higman, et al., J. Biol. Chem. 267: 16430, 1992).

The following enzymatic reactions are involved in capping of eukaryoticmRNA:

(1) RNA triphosphatase cleaves the 5′-triphosphate of mRNA to adiphosphate,

pppN₁(p)N_(x)-OH(3′)→ppN₁(pN)_(x)-OH(3′)+Pi; and then

(2) RNA guanyltransferase catalyzes joining of GTP to the 5′-diphosphateof the most 5′ nucleotide (N₁) of the mRNA,

ppN₁(pN)_(x)-OH(3′)+GTP→G(5′)ppp(5′)N₁(pN)_(x)-OH(3′)+PPi; and finally,

(3) guanine-7-methyltransferase, using S-adenosyl-methionine (AdoMet) asa co-factor, catalyzes methylation of the 7-nitrogen of guanine in thecap nucleotide,

G(5′)ppp(5′)N₁(pN)_(x)-OH(3′)+AdoMet→m⁷G(5′)ppp(5′)N₁(pN)_(x)-OH(3′)+AdoHyc.

RNA that results from the action of the RNA triphosphatase and the RNAguanyltransferase enzymatic activities, as well as RNA that isadditionally methylated by the guanine-7-methyltransferase enzymaticactivity, is referred to as “5′ capped RNA” or “capped RNA”, and thecombination of one or more polypeptides having the enzymatic activitiesthat result in “capped RNA” are referred to as “capping enzyme systems”or, more simply, as “capping enzymes” herein. Capping enzyme systems,including cloned forms of such enzymes, have been identified andpurified from many sources and are well known in the art (e.g., seeShuman, S, Prog. Nucleic Acid Res. Mol. Biol. 66: 1-40, 2001; Shuman, S,Prog. Nucleic Acid Res. Mol. Biol. 50: 101-129, 1995; and Banerjee, A K,Microbiol. Rev. 44: 175, 1980). The capped RNA that results from theaddition of the cap nucleotide to the 5′-end of primary RNA by a cappingenzyme system has been referred to as capped RNA having a “cap 0structure” (e.g., see Higman, M A et al., J. Biol. Chem. 269:14974-14981, 1994; Myette, J R and Niles, E G, J. Biol. Chem. 271:11936-11944, 1996). Capping enzyme systems have been used to synthesizecapped RNA having a cap 0 structure in vitro (e.g., see Shuman, S etal., J. Biol. Chem. 255: 11588, 1980; Wang, S P et al., Proc. Natl.Acad. Sci. USA 94: 9573, 1997; Higman M. A. et al., J. Biol. Chem. 267:16430, 1992; Higman M. A. et al., J. Biol. Chem. 269: 14974, 1994;Myette, J. R. and Niles, E. G., J. Biol. Chem. 271: 11936, 1996; andreferences therein).

Capped RNA having a cap 0 structure can be further transformed in vivoor in vitro to a “cap I” structure by the action of an enzyme with mRNA(nucleoside-2′-O—) methyltransferase activity (e.g., see Higman, M A etal., J. Biol. Chem. 269: 14974-14981, 1994; Myette, J R and Niles, E G,J. Biol. Chem. 271: 11936-11944, 1996). A capped RNA with a “cap I”structure, in addition to having a 7-methyl-G cap nucleotide as the 5′ultimate cap nucleotide, also has a 2′-O-methyl group on the5′-penultimate nucleotide. For example, vaccinia mRNA (nucleoside-2′-O)methyltransferase can catalyze methylation of the 2′-hydroxyl group ofthe 5′-penultimate nucleotide of 5′-capped RNA having a cap 0 structure,as follows:

m⁷G(5′)ppp(5′)N₁(pN)_(x)-OH(3′)+AdoMet→m⁷G(5′)ppp(5′)m^(2′-O)N₁(pN)_(x)-OH(3′)+AdoHyc.

Dimethylated capped RNAs having a cap I structure have been reported tobe translated more efficiently than 7-methylguanosine-capped RNAs havinga cap 0 structure (e.g., see Kuge, H et al., Nucleic Acids Res. 26:3208, 1998).

Most commonly, the RNA that has been used for in vitro capping reactionshas been obtained using a T7, T3 or SP6 RNA polymerase for in vitrotranscription of a template that is downstream of the respective RNApolymerase promoter, but primary RNA from other sources can also beused.

During the 1970s and early 1980s, capping enzymes were used as reagentsfor capping of RNAs. However, in the mid-1980s, this method forsynthesis of capped transcripts was supplanted by the use of adinucleotide cap analog to prime in vitro transcription with phage RNApolymerases (Melton, D et al., Nucleic Acids Res. 12: 7035, 1984). Sincethat time, post-transcriptional capping of RNA in an in vitro reactionusing a capping enzyme system has not been widely used except bylaboratories studying capping enzymes, and the most frequently used invitro method to make capped RNAs having a cap 0 structure has beentranscription of a DNA template with either a bacterial RNA polymeraseor a bacteriophage RNA polymerase in the presence of all fourribonucleoside triphosphates and a “dinucleotide cap analog”, alsoreferred to as a “cap analog.” A cap analog, such as m⁷G(5′)ppp(5′)G(also referred to as “m⁷GpppG”), is a dinucleotide consisting of anouter cap nucleoside, such as 7-methyl-guanosine (m⁷G), and thenucleotide corresponding to the first nucleotide of the primarytranscript (e.g., in this case, G). This cap analog is often usedbecause the primary nucleotide (i.e., the most 5′ nucleotide) of most,but not all, primary RNA transcripts synthesized using phage RNApolymerase transcription systems is guanosine ribonucleotide.

Most commonly, capped RNAs are synthesized using this method bycell-free transcription of DNA templates (e.g., Contreras, R. et al.,Nucl. Acids Res. 10: 6353, 1982; Yisraeli J. et al., Meth. Enzymol. 180:42-50, 1989; and Melton, D. et al., Nucl. Acids Res. 12: 7035-7056,1984). When capping is carried out using a cap analog in such an invitro transcription reaction, the RNA polymerase initiates transcriptionby extension of the 3′-OH of the cap analog, rather than by extension ofthe 3′-OH of an initiating nucleoside triphosphate. Thus, if the m⁷GpppGcap analog is used, the initial product is expected to be m⁷GpppGpN. Thealternative, GTP-initiated product pppGpN is suppressed by setting theratio of m⁷GpppG to GTP between about 4-to-1 to about 10-to-1 in thetranscription reaction mixture.

However, when using a cap analog in an in vitro transcription reactionto make capped RNA, Pasquinelli, A. et al. (RNA 1: 957-967, 1995) foundthat, in addition to obtaining the expected m⁷GpppGpN product,approximately one-third to one-half of the capped RNA products made withthis m⁷GpppG cap analog actually had the “reverse cap” Gpppm⁷GpN,demonstrating that bacteriophage RNA polymerases can also use the 3′-OHof the 7-methylguanosine moiety of m⁷GpppG to initiate transcription.Such reverse-capped RNA molecules behaved abnormally. For example,Pasquinelli et al. reported that when reverse-capped pre-U1 RNAtranscripts were injected into Xenopus laevis nuclei, they were exportedmore slowly than natural transcripts. Similarly, cytoplasmicreverse-capped U1 RNAs in the cytoplasm were not properly imported intothe nucleus. Because the resulting capped RNAs contain about one-thirdto one-half reverse caps, the overall translational activity of such invitro-synthesized mRNA is reduced and other functional properties of themRNA may also be affected. Thus, translation of in vitro-synthesizedmRNAs having such reverse caps is impaired.

To address the problem of dinucleotide cap analogs being incorporated inthe reverse orientation during in vitro transcription reactions,Stepinski et al. (Nucleosides and Nucleotides 14: 717-721, 1995) andPeng et al. (Organic Letters 4: 161-164, 2002) synthesized dinucleotidecap analogs which could only be incorporated in the correct orientationbecause the 3′-OH of the cap nucleotide was eliminated or blocked bysubstitution. Since they could not be incorporated in the reverseorientation, Stepinski et al. referred to these dinucleotide cap analogsas “anti-reverse cap analogs” or “ARCAs”. Using RNA transcripts made invitro in the presence of several different ARCAs, including m₂^(7, 3′-O)GpppG, it has been demonstrated that ARCA-capped RNAs resultin higher translational efficiencies than RNA transcripts made in thepresence of the standard m⁷GpppG cap analog, both for RNA transcriptstranslated in a rabbit reticulocyte lysate in vitro (Stepinski et al.,Nucleosides and Nucleotides 14: 717-721, 1995; U.S. Patent ApplicationNo. 200301945759; Jemielity et al., RNA 9: 1108-1122, 2003; Grudzien etal., RNA 10: 1479-1487; 2004) and for RNA transcripts electroporatedinto mouse mammary epithelial (MM3MG) cells and translated in vivo(Grudzien et al., J. Biol. Chem. 281: 1857-1867, 2006). Mockey et al.(Biochem. Biophys. Res. Comm. 340: 1062-1068, 2006) also found thatlipofection of mouse dendritic cells with a luciferase mRNA having a3′-poly(A) tail of defined length was translated with higher efficiencyif the mRNA used was capped using the m₂ ^(7, 3′-O) GpppG ARCA than ifit was capped using the standard m⁷GpppG cap analog. The dinucleotidecap analog m₂ ^(7, 2′-O) GpppG is also incorporated only in the correctorientation and is therefore an ARCA (Jemielity et al., RNA 9:1108-1122, 2003; Grudzien et al., RNA 10: 1479-1487; 2004). RNA cappedwith m₂ ^(7, 2′-O) GpppG was also translated in vitro with higherefficiency than the standard m⁷GpppG cap analog (Jemielity et al., RNA9: 1108-1122, 2003; Grudzien et al., RNA 10: 1479-1487; 2004). Thus,mRNA having a cap nucleotide that is methylated in the 2′ or 3′-positionwas beneficial for improving translational efficiency of mRNA in vitroand in vivo.

However, although RNA can be capped by in vitro transcription of a DNAtemplate in the presence of an ARCA, this approach has severaldrawbacks. First, the chemical syntheses of ARCAs (e.g., see Jemielity,J et al., RNA: 1108, 2003) are difficult (˜6 synthetic steps),time-consuming (˜12 weeks) and expensive. Also, once the ARCA isobtained, the in vitro transcription reaction is wasteful andinefficient. Due to the limiting amount of GTP in the reaction (since80% or more of the GTP is typically substituted by ARCA), the RNA yieldof the in vitro transcription with a cap analog is at best 33% of theRNA yield obtained without cap analog. Not only is the RNA yield lowerusing a cap analog, but also <80% of the RNA obtained is capped. Stillfurther, the fact that the cap analog can never be incorporated to 100%limits the purity of the capped RNA product, necessitating more work topurify the product and increasing the risk that the capped RNA productwill still be contaminated with impurities, including unincorporated capanalog. This is particularly detrimental if the capped RNA is to be usedfor medical applications, such as for therapeutics, or for clinicalresearch, since the contaminants may produce undesired effects. Thus,there is a need for compositions and methods that provide consistent 5′capping of RNA in a correct orientation and that increase incorporationefficiency, such as in order to improve the stability of invitro-generated RNA transcripts (e.g., thereby increasing translationalefficiency).

Also, little was known about the possibility of using a modified capnucleotide as a substrate for a capping enzyme system to make RNA withimproved properties. Published work related to use of a modifiednucleotide as a substrate for a capping enzyme discouraged thisapproach. For example, the data of Shuman et al. (J. Biol. Chem. 255:11588, 1980) indicated that only GTP was a good substrate for the RNAguanyltransferase activity of the vaccinia capping enzyme system; UTP,CTP, ATP, ITP, GDP, GMP and N⁷-methyl-GTP could not be used in place ofGTP in an in vitro capping reaction. They found that 2′-dGTP seemed tohave slight activity in a [³²P]-PPi exchange assay in the presence ofpermeabilized vaccinia virions, indicating that it might be a substratefor capping, but it was only approximately 6% as active as GTP in thisassay. Thus, 2′-dGTP appeared to be incorporated into capped RNA, butinefficiently. Bougie, I and Bisaillon, M (J. Biol. Chem. 279: 22124,2004) found that the intracellular triphosphate metabolite of theantiviral nucleoside ribavirin, a nucleoside with a monocyclic baseanalog, was of ribavirin, was a substrate for viral capping enzyme; theribavirin-capped RNA was more stable than uncapped RNA, but was not afunctional mimic of the N⁷-methyl-guanosine cap with respect totranslation (Yan, Y et al., RNA 11: 1238-1244, 2005; Westman, B et al.,RNA 11: 1505-1513, 2005).

It would be highly desirable if there was an easier, faster, lessexpensive, higher yield way to make capped RNA transcripts, includingmodified nucleotide-capped RNA transcripts, particularly transcriptsthat are of a higher purity, for a variety of applications, includingmedical applications.

Still further, in vitro transcription in the presence of an ARCA resultsin RNA having a cap 0 structure. However, a capped RNA with a cap Istructure could not be synthesized using an m⁷Gpppm^(2′-O) G cap analog(Pasquinelli, RNA 1: 957, 1995). Also, ARCA-capped RNA has not been usedas a substrate for methylation of the 5′-penultimate nucleotide of thecapped RNA using mRNA (nucleoside-2′-O—) methyltransferase to obtaincapped RNA having a cap I structure, which is unfortunate because2′-O-methylation has been shown to significantly enhance translationcompared to capped RNA having a type 0 structure. Therefore, it wouldalso be highly desirable if there was a way to makemodified-nucleotide-capped RNA having a cap I structure for a variety ofapplications, including medical applications.

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

“Affinity binding molecules” or “specific binding pair” herein means twomolecules that have affinity for and “bind” to each other under certainconditions, referred to as “binding conditions. Biotin and streptavidinor avidin are examples of a “specific binding pair” or “affinity bindingmolecules”, but the invention is not limited to use of this particularspecific binding pair. In many embodiments of the present invention, onemember of a particular specific binding pair is referred to as the“affinity tag molecule” or the “affinity tag” and the other as the“affinity-tag-binding molecule” or the “affinity tag binding molecule”.For example, but without limitation, in some embodiments, biotin isreferred to as the affinity tag or affinity tag molecule, and astreptavidin or avidin molecule, whether it is free, attached to asurface, attached to another molecule, or labeled with a detectablemolecule such as a dye, is referred to as the affinity-tag-bindingmolecule. In other embodiments, streptavidin is the affinity tag andbiotin is the affinity-tag-binding molecule, since streptavidin andbinding function together as a specific binding pair or as affinitybinding molecules. A wide variety of other specific binding pairs oraffinity binding molecules, including both affinity tag molecules andaffinity-tag-binding molecules, are known in the art (e.g., see U.S.Pat. No. 6,562,575), which can be used in the present invention. Forexample, an antigen (which itself may be an antibody) and an antibody,including a monoclonal antibody, that binds the antigen is a specificbinding pair. Also, an antibody and an antibody binding protein, such asStaphylococcus aureus Protein A, can be employed as a specific bindingpair. Other examples of specific binding pairs include, but are notlimited to, a carbohydrate moiety which is bound specifically by alectin and the lectin; a hormone and a receptor for the hormone; and anenzyme and an inhibitor of the enzyme. Usually, molecules that comprisea specific binding pair interact with each other only throughnon-covalent bonds such as hydrogen-bonding, hydrophobic interactions(including stacking of aromatic molecules), van der Waals forces, andsalt bridges. Without being bound by theory, it is believed in the artthat these kinds of non-covalent bonds result in binding, in part due tocomplementary shapes or structures of the molecules involved in thebinding pair. The term “binding” according to the invention refers tothe interaction between an affinity binding molecules or specificbinding pairs (e.g., between biotin as an affinity tag molecule andstreptavidin as an affinity-tag-binding molecule) as a result ofnon-covalent bonds, such as, but not limited to, hydrogen bonds,hydrophobic interactions, van der Waals bonds, and ionic bonds. Based onthe definition for “binding,” and the wide variety of affinity bindingmolecules or specific binding pairs, it is clear that “bindingconditions” vary for different specific binding pairs. Those skilled inthe art can easily determine conditions whereby, in a sample, bindingoccurs between the affinity binding molecules. In particular, thoseskilled in the art can easily determine conditions whereby bindingbetween affinity binding molecules that would be considered in the artto be “specific binding” can be made to occur. As understood in the art,such specificity is usually due to the higher affinity between theaffinity binding molecules than for other substances and components(e.g., vessel walls, solid supports) in a sample. In certain cases, thespecificity might also involve, or might be due to, a significantly morerapid association of affinity binding molecules than with othersubstances and components in a sample.

In some embodiments of the invention, an “affinity tag reagent” or and“affinity tag having a reactive moiety” is used, by which we meanherein, a molecule that comprises both an affinity tag and a reactivechemical group or moiety that is capable of reacting with one or moreatoms or groups of the molecule with which it reacts to form one or morecovalent chemical bonds between the molecule comprising the affinity tagand the molecule with which it reacts. By way of example, but withoutlimitation, in some embodiments, the affinity tag reagent is anacylating reagent (e.g., an N-hydroxysuccinimidyl ester), wherein theaffinity tag is chemically joined to an atom in the molecule with whichit reacts via an acyl linkage. In other embodiments, the affinity tagreagent is an alkylating reagent, group, wherein the affinity tag ischemically joined to an atom in the molecule with which it reacts via analkyl linkage. In other embodiments, the affinity tag reagent reacts viaan electrocyclic type of chemical reaction, such as a 1,3-dipolarcycloaddition (e.g., cycloaddition of an alkyne with an azide). Thus,the term “reactive” moiety with respect to, for example, an “affinitytag reagent” or “affinity tag having a reactive moiety” is used to referto a moiety or group that is involved in or responsible for the chemicalreaction whereby a molecule comprising the affinity tag reactschemically to form a covalent chemical bond with one or more atoms inthe molecule with which it reacts, rather than to the binding thatresults between affinity binding molecules due to non-covalent forcesand bonds.

When we refer to attaching the “affinity-tag-binding molecule” or the“affinity tag binding molecule”, such as streptavidin or avidin,directly to the surface, we usually, but not always mean that theaffinity-tag-binding molecule is covalently attached to the surface bymeans of a chemical linker that is joined to the surface and to theaffinity-tag-binding molecule. When we refer to attaching the“affinity-tag-binding molecule” or the “affinity tag binding molecule”,such as streptavidin or avidin, indirectly to the surface, we mean thatthe affinity-tag-binding molecule is bound to another molecule withwhich it has affinity (e.g., an anti-streptavidin antibody) that is inturn bound to the surface. In some embodiments, the affinity-tag-bindingmolecule, such as streptavidin or avidin, is not attached to a surface,but is bound by another molecule, such as an antibody or Protein A, andthe biotinylated modified-nucleotide-capped RNA is recovered byprecipitation or by binding to a second antibody or other molecule usingmethods and compositions known in the art.

A “cap nucleotide” of the present invention means anucleoside-5′-triphosphate that, under suitable reaction conditions, isused as a substrate by a capping enzyme system and that is therebyjoined to the 5′-end of an uncapped RNA comprising primary RNAtranscripts or RNA having a 5′-diphosphate. The nucleotide that is sojoined to the RNA is also referred to as a “cap nucleotide” herein.

A “capping enzyme system” or a “capping enzyme” of the present inventionmeans the combination of one or more polypeptides having enzymaticactivities that, in the presence of a cap nucleotide, including amodified cap nucleotide, and suitable reaction conditions, results insynthesis of capped RNA, including a modified-nucleotide-capped RNA,having a cap 0 structure. In general, a capping enzyme system or cappingenzyme of the invention comprises RNA triphosphatase and RNAguanyltransferase enzymatic activities, and optionally, the cappingenzyme system can also comprise RNA guanine-7-methyltransferaseenzymatic activity. Capping enzyme systems that can be used for thepresent invention are well known in the art (e.g., see Shuman, S, Prog.Nucleic Acid Res. Mol. Biol. 66: 1-40, 2001; Shuman, S, Prog. NucleicAcid Res. Mol. Biol. 50: 101-129, 1995; Bisaillon, M and Lemay, G,Virology 236: 1-7, 1997; Banerjee, A K, Microbiol. Rev. 44: 175-205,1980). Without limiting the invention, vaccinia virus capping enzyme andpoxvirus capping enzymes having these enzymatic activities are known inthe art that can be used as a capping enzyme system of the invention,including both full-length and enzymatically active portions thereof,which capping enzymes have been identified, purified, characterized,cloned, and expressed from a clone (e.g., see Martin S A et al., J BiolChem 250: 9322-9329, 1975; Shuman, J Biol Chem 265: 11960-11966, 1990;Shuman and Morham, 265: 11967-11972, 1990; M A Higman, et al., J. Biol.Chem. 267: 16430, 1992; Myette, J R and Niles, E G, J. Biol. Chem. 271:11936, 1996). Thus, in some embodiments, the capping enzyme, such asvaccinia virus capping enzyme or capping enzyme encoded by a poxviruscapping enzyme gene known in the art, is purified from a clone thatexpresses a nucleic acid or polynucleotide sequence encoding thefull-length gene or an enzymatically active portion thereof. The presentinvention is not limited by the type of capping enzyme utilized. Forexample, in some embodiments in which vaccinia virus capping enzyme isused, the capping enzyme is purified from vaccinia virus, whereas inother embodiments, the vaccinia capping enzyme is a purified recombinantvaccinia virus capping enzyme. In some embodiments, the vaccinia viruscapping enzyme is a mutant or variant of the wild-type enzyme (e.g.,that exhibits greater enzymatic activity compared to wild-type cappingenzyme). Variants, including allelic variants, muteins, analogs andfragments capable of functioning as the provided capping enzyme areknown in the art and are also contemplated by this invention. The activesites for the RNA triphosphatase, RNA guanyltransferase andguanine-7-methyltransferase enzymatic activities can be onsingle-component polypeptides, 2-component polypeptides (typicallyhaving RNA triphosphatase and RNA guanyltransferase activities), or on a3-component polypeptide, from a cloned or a wild-type source. In view ofthe fact that genes encoding RNA triphosphatase, RNA guanyltransferaseand guanine-7-methyltransferase from one source can complement deletionsin one or all of these genes from another source (e.g., see reference tounpublished work of Schwer et al. on p. 3 of Shuman, S, Prog. NucleicAcid Res. Mol. Biol. 66: 1-40, 2001 with respect to Saccharomycescerevisiae, Schizosaccharomyces pombe and Candida albicans), the cappingenzyme system can originate from one wild type source, or one or more ofthe RNA triphosphatase, RNA guanyltransferase, and/orguanine-7-methyltransferase activities can comprise a polypeptide from adifferent source, which polypeptides can each be encoded by a DNAsequence originating from the same biological source or by a DNAsequence originating from a different biological source. In preferredembodiments, the RNA triphosphatase component of the capping enzymecomprises a divalent cation-dependent RNA triphosphatase encoded by aDNA virus or fungus having conserved motifs A, B, and C. In somepreferred embodiments, the RNA triphosphatase is encoded by a poxvirusgene. In some preferred embodiments, the RNA triphosphatase is encodedby a vaccinia virus gene. However, the invention is not limited to adivalent cation-dependent RNA triphosphatase encoded by a DNA virus orfungus having conserved motifs A, B, and C (see Shuman, S, Prog. NucleicAcid Res. Mol. Biol. 66: 1-40, 2001); in some embodiments the RNAtriphosphatase comprises a divalent cation-independent RNAtriphosphatase encoded by a DNA derived from a nematode, mammalian orother metazoan source, so long as the RNA triphosphatase removes a gammaphosphate of a triphosphate-terminated RNA to form an RNA with a5′-diphosphate terminus. In view of the fact that vaccinia virus RNAtriphosphatase-defective mutants can transfer GMP to 5′-triphosphate RNAends to produce a cap with a tetraphosphate linkage (Yu, L and Shuman,S, J. Virology 70: 6162-6168, 1996) and that RNA with tetraphosphatecaps can be translated with good efficiency (e.g., Grudzien et al., RNA10: 1479, 2004), the capping enzyme system can lack RNA triphosphataseactivity in some embodiments. RNA guanyltransferases of capping enzymesystems are structurally and mechanistically conserved among fungi,metazoans, protozoa, and DNA viruses (Shuman, S, Prog. Nucleic Acid Res.Mol. Biol. 66: 1-40, 2001). In some embodiments of the invention, thecapping enzyme has a conserved motif I consisting of the amino acidsequence KxDGxx (SEQ ID NO:1). In some embodiments, the sixth (6th)position of said motif I is not arginine. Motif I contains the activesite of covalent attachment of GMP to the capping enzyme within the RNAguanyltransferase portion of capping enzyme. In some embodiments, motifI of the capping enzyme has the amino acid sequence KTDG(I/V)(P/G) (SEQID NO:2). In some embodiments, motif I of the capping enzyme has theamino acid sequence KTDG(I/V)x (SEQ ID NO:3), wherein the 6th amino acidof said motif I is an amino acid selected from the group consisting ofphenylalanine, serine, and leucine. Another conserved motif within thecapping enzyme is motif III, which is also within the RNAguanyltransferase portion of the capping enzyme. In some embodiments,the first position of motif III is valine, isoleucine, or tyrosine, thesixth position of motif III is glutamic acid, and the fourth position ofmotif III is phenylalanine, tyrosine or tryptophan. In one embodiment ofthe capping enzyme, motif III has the amino acid sequence VVVFGEAV (SEQID NO:4). In some embodiments, motif III has the amino acid sequenceYRLWCEAV (SEQ ID NO:5). In some embodiments, motif III has the aminoacid sequence VT(L/I)YGEA(I/V) (SEQ ID NO:6). In some embodiments, motifIII has the amino acid sequence (V/I)YLYAEMR (SEQ ID NO:7). In someembodiments, motif III has the amino acid sequence (V/I)xL(Y/F)GEA(I/V)(SEQ ID NO:8). In some embodiments, the RNA guanyltransferase is encodedby a poxvirus gene. In some embodiments, the RNA guanyltransferase isencoded by a vaccinia virus gene. Whereas the RNA guanyltransferasereaction step is reversible, the methylation step catalyzed by theguanine-7-methyltransferase activity of the capping enzyme isessentially irreversible. Therefore, 7-methylation of the guanine isuseful because this step drives the reaction to completion in thedirection of cap formation. Methylation of the cap is also useful forenhancing translation of the RNA. The amino acid sequence and structureof guanine-7-methyltransferase enzymes of capping enzymes are highlyconserved from DNA viruses to yeast to humans and other metazoans. Thisis shown by the fact that full-length and some truncatedguanine-7-methyltransferase genes that encode S. pombe, C. albicans andhuman capping enzymes can complement deletions of the S. cerevisiaecapping enzyme guanine-7-methyltransferase, by the fact that the guanine7-methyltransferase domain of vaccinia virus capping enzyme can functionin vivo in lieu of the yeast methyltransferase enzyme (Saha, N et al.,J. Virology 77: 7300-7307, 2003), and also by the concordance ofmutational effects between the yeast, human and vaccinia enzymes(Shuman, S, Prog. Nucleic Acid Res. Mol. Biol. 66: 1-40, 2001).Therefore, the guanine-7-methyltransferase portion of a capping enzymeof the invention can comprise a wild-type or recombinant enzyme from anyof a wide variety of sources. In some embodiments, theguanine-7-methyltransferase is encoded by a poxvirus gene. In someembodiments, the guanine-7-methyltransferase is encoded by a vacciniavirus gene. In some embodiments, the guanine 7-methyltransferase has aconserved IHF amino acid motif. In some embodiments, the guanine7-methyltransferase has a motif consisting of the amino acid sequenceVL(D/E)xGxGxG (SEQ ID NO:9).

By “condition-specific” RNA is meant an RNA sample that, relative tounfractionated condition-derived RNA, has a higher content of RNA thatis preferentially present in the condition-specific cell compared with acell without the condition, wherein a “condition” means a mode or stateof being of the organism or cells from which the biological sample isderived (e.g., a cancer condition versus a non-cancerous condition, or apathogen-infected condition versus an uninfected condition). Forexample, but without limitation, in some embodiments, the subtracted andamplified RNA can comprise RNA from a condition (e.g., a tumor cellcondition) from which RNA that is also present in a normal cell of thesame type has been removed by subtractive hybridization and digestion,and then the remaining RNA is amplified using an RNA amplificationreaction. In some embodiments of the invention, the uncapped RNAcomprising primary RNA transcripts or RNA having a 5′-diphosphate usedfor synthesizing the modified-nucleotide-capped RNA comprises primaryRNA transcripts or RNA having a 5′-diphosphate obtained using thesubtractive hybridization and digestion, and RNA amplification methodsdescribed in U.S. Pat. No. 5,712,127. In some embodiments, the uncappedRNA comprising primary RNA transcripts or RNA having a 5′-diphosphateused for synthesizing the modified-nucleotide-capped RNA is obtainedusing the “Cap-Dependent Subtraction” (“CDS”) method of the presentinvention described herein. In some embodiments, the RNA amplificationmethod is an RNA amplification reaction or method as defined herein,such as, but not limited to the terminal tagging method described inU.S. Patent Application No. 20050153333 of Sooknanan.Modified-nucleotide-capped RNA made using the uncapped RNA comprisingprimary RNA transcripts or RNA having a 5′-diphosphate from suchsubtractive hybridization and digestion, and RNA amplification reactionsis referred to as “condition-specific” (e.g., “tumor-specific”), and, ifthe modified-nucleotide-capped RNA is subsequently used to synthesizepolypeptides (or antigenic epitopes of the polypeptides) by in vitrotranslation or used to transfect a cell, wherein themodified-nucleotide-capped RNA is translated in vivo, the invitro-synthesized polypeptides or the polypeptides expressed in vivo inthe cells are also called “condition-specific.” Similarly, any responsesthat are expressed in any other cells or organisms that are exposed tothe condition-specific modified-nucleotide-capped RNA orcondition-specific polypeptides, antigens, or immune responses derivedtherefrom are referred to as “condition-specific.” For example, butwithout limitation, an antigen-presenting cell (APC) that is loaded witha tumor-specific modified-nucleotide-capped RNA or polypeptidestranslated therefrom, the APC cell (and any antigenic epitopes presentedon its surface) is referred to as tumor-specific. The invention is notlimited by the particular condition. For example, but withoutlimitation, in some embodiments, the uncapped RNA is derived from acondition comprising a tumor or cancer condition and thecondition-specific uncapped RNA is referred to as “tumor-specific” or“cancer-specific.” In other embodiments, the uncapped RNA is derivedfrom a pathogen, such as a bacterial, viral or fungal pathogen, or froma eukaryotic cell that is infected by a bacterial, viral or fungalpathogen, and the uncapped RNA is referred to as “pathogen-specific.”

A “modified cap nucleotide” of the present invention means a capnucleotide comprising: (a) a modified 2′- or3′-deoxyguanosine-5′-triphosphate, wherein the 2′- or 3′-deoxy positionof the sugar moiety is substituted by a group other than a hydroxylgroup or a hydrogen, or wherein the O6 oxygen of the guanine base isreplaced by a thiol (or mercapto) group or a methylthio (ormethylmercapto) group; or (b) a modified guanosine-5′-triphosphate,wherein the 2′- or 3′-hydroxyl group of the ribose is substituted by analkyl group or, wherein the N1 nitrogen or the O6 oxygen of the guaninebase is substituted by an alkyl group or, wherein the O6 oxygen of theguanine base is replaced by a thiol (or mercapto) group or a methylthio(or methylmercapto) group; or (c) 3′-deoxyguanosine. In some preferredembodiments of the invention, the modified cap nucleotide comprises: (i)a modified 2′- or 3′-deoxyguanosine-5′-triphosphate (or guanine 2′- or3′-deoxyribonucleic acid-5′-triphosphate) wherein the 2′- or 3′-deoxyposition of the deoxyribose sugar moiety is substituted with a groupcomprising an amino group, an azido group, a fluorine group, a methoxygroup, a thiol (or mercapto) group or a methylthio (or methylmercapto)group; or (ii) a modified guanosine-5′-triphosphate, wherein the N1nitrogen or the O6 oxygen of the guanine base is substituted with amethyl group, or wherein the O6 oxygen is replaced by a thiol (ormercapto) group or methylthio (or methylmercapto) group; or (iii)3′-deoxyguanosine. For the sake of clarity, it will be understood hereinthat an “alkoxy-substituted deoxyguanosine-5′-triphosphate” can also bereferred to as an “O-alkyl-substituted guanosine-5′-triphosphate”; byway of example, but without limitation,2′-methoxy-2′-deoxyguanosine-5′-triphosphate (2′-methoxy-2′-dGTP) and3′-methoxy-3′-deoxyguanosine-5′-triphosphate (3′-methoxy-3′-dGTP) canalso be referred to herein as 2′-O-methylguanosine-5′-triphosphate(2′-OMe-GTP) and 3′-O-methylguanosine-5′-triphosphate (3′-OMe-GTP),respectively. Following joining of the modified cap nucleotide to the5′-end of the uncapped RNA comprising primary RNA transcripts or RNAhaving a 5′-diphosphate, the portion of said modified cap nucleotidethat is joined to the uncapped RNA comprising primary RNA transcripts orRNA having a 5′-diphosphate is usually referred to herein as a “modifiedcap nucleoside” (i.e., without referring to the phosphate groups towhich it is joined), but sometimes it is referred to herein as a“modified cap nucleotide”.

A “modified-nucleotide-capped RNA” of the present invention is a cappedRNA molecule that is synthesized using a capping enzyme system and amodified cap nucleotide, wherein the cap nucleotide on its 5′ terminuscomprises the modified cap nucleotide.

As used herein, “poxviruses” (or Poxviridae) means a member of a familyof brick-shaped or ovoid viruses that contains a double-stranded DNAgenome. For example, but without limitation, poxviruses include vacciniavirus, variola virus, rabbitpox virus, monkeypox virus, ectromeliavirus, camelpox virus, cowpox virus, muledeerpox virus, myxoma virus,rabbit fibroma virus, swinepox virus, lumpy skin disease virus, sheeppoxvirus, canarypox virus, fowlpox virus, orf virus, and bovine papularstomatitis virus, as well as relatives, descendents, variants, andderivatives of such poxviruses.

A “primary RNA” or “primary RNA transcript” means the RNA molecule thatis newly synthesized by an RNA polymerase in vivo or in vitro and whichRNA molecule has a triphosphate on the 5′-carbon of its most 5′nucleotide.

“Transcription” means the formation or synthesis of an RNA molecule byan RNA polymerase using a DNA molecule as a template.

“Replication” means the formation or synthesis of an RNA molecule by anRNA-dependent RNA polymerase (or “replicase”) using an RNA molecule as atemplate.

An “RNA amplification reaction” or an “RNA amplification method” means amethod for increasing the amount of RNA corresponding to one or multipledesired RNA sequences in a sample. For example, in some embodiments, theRNA amplification method comprises: (a) synthesizing first-strand cDNAcomplementary to the one or more desired RNA molecules by RNA-dependentDNA polymerase extension of one or more primers that anneal to thedesired RNA molecules; (b) synthesizing double-stranded cDNA from thefirst-strand cDNA using a process wherein a functional RNA polymerasepromoter is joined thereto; and (c) contacting the double-stranded cDNAwith an RNA polymerase that binds to said promoter under transcriptionconditions whereby RNA corresponding to the one or more desired RNAmolecules is obtained. Unless otherwise stated related to a specificembodiment of the invention, an RNA amplification reaction according tothe present invention means a sense RNA amplification reaction, meaningan RNA amplification reaction that synthesizes sense RNA (e.g., RNAhaving the same sequence as an mRNA or other primary RNA transcript,rather than the complement of that sequence). Sense RNA amplificationreactions known in the art, which are encompassed within this definitioninclude, but are not limited to, the methods which synthesize sense RNAdescribed in U.S. Patent Application No. 20050153333 of Sooknanan; U.S.Patent Application No. 20030186237 of Ginsberg, Stephen; U.S. PatentApplication No. 20040197802 of Dahl and Jendrisak; and U.S. PatentApplication No. 20040171041 of Dahl et al, and in Ozawa, T et al.(Biotechniques 40: 469-478, 2006).

A “poly(A) polymerase” (“PAP”) means a template-independent RNApolymerase found in most eukaryotes, prokaryotes, and eukaryotic virusesthat selectively uses ATP to incorporate AMP residues to 3′-hydroxylatedends of RNA. Since PAP enzymes that have been studied from plants,animals, bacteria and viruses all catalyze the same overall reaction(e.g., see Edmonds, M, Methods Enzymol., 181; 161-180, 1990), are highlyconserved structurally (e.g., see Gershon, P, Nature Structural Biol. 7:819-821, 2000), and lack intrinsic specificity for particular sequencesor sizes of RNA molecules if the PAP is separated from proteins thatrecognize AAUAAA polyadenylation signals (Wilusz, J and Shenk, T, Cell52: 221, 1988), purified wild-type and recombinant PAP enzymes from anyof a variety of sources can be used in the kits and methods of thepresent invention.

As used herein, a “T7-type” RNA polymerase (RNAP) means T7 RNApolymerase (e.g., see Studier, F W et al., pp. 60-89 in Methods inEnzymology, Vol. 185, ed. by Goeddel, D V, Academic Press, 1990) or anRNAP derived from a “T7-type” bacteriophage, meaning a bacteriophagethat has a similar genetic organization to that of bacteriophage T7.Examples of T7-type bacteriophages according to the invention include,but are not limited to Escherichia coli phages T3, phi I, phi II, W31,H, Y, A1, 122, cro, C21, C22, and C23; Pseudomonas putida phage gh-1;Salmonella typhimurium phage SP6; Serratia marcescens phages IV;Citrobacter phage ViIII; and Klebsiella phage No. 11 (Hausmann, CurrentTopics in Microbiology and Immunology 75:77-109, 1976; Korsten et al.,J. Gen. Virol. 43:57-73, 1975; Dunn, et al., Nature New Biology230:94-96, 1971; Towle, et al., J. Biol. Chem. 250:1723-1733, 1975;Butler and Chamberlin, J. Biol. Chem. 257:5772-5778, 1982), as well asmutant forms of such RNAPs (e.g., Sousa et al., U.S. Pat. No. 5,849,546;Padilla, R and Sousa, R, Nucleic Acids Res., 15: e138, 2002; Sousa, Rand Mukherjee, S, Prog Nucleic Acid Res Mol Biol., 73: 1-41, 2003).

With respect to the use of the word “derived”, such as for an RNA(including a modified-nucleotide-capped RNA) or a polypeptide that is“derived” from a condition, biological sample, sample, tumor, pathogen,or the like, it is meant that the RNA or polypeptide either was presentin the condition, biological sample, sample, tumor, or pathogen, or wasmade using the RNA in the condition, biological sample, sample, tumor,or pathogen by a process such as, but not limited to, an RNAamplification reaction, wherein the RNA or polypeptide is either encodedby or a copy of all or a portion of the RNA or polypeptide molecules inthe original condition, biological sample, sample, tumor, or pathogen.By way of example, but without limitation, such RNA can be from an invitro transcription or an RNA amplification reaction, with or withoutcloning of cDNA, rather than being obtained directly from the condition,biological sample, sample, tumor, or pathogen, so long as the originalRNA used for the in vitro transcription or an RNA amplification reactionwas from the condition, biological sample, sample, tumor, or pathogen.

As used herein, the term “purified” or “to purify” refers to the removalof components (e.g., contaminants) from a sample. For example, nucleicacids are purified by removal of contaminating cellular proteins orother undesired nucleic acid species. The removal of contaminantsresults in an increase in the percentage of desired nucleic acid in thesample.

As used herein, the term “in vitro” refers to an artificial environmentand to processes or reactions that occur within an artificialenvironment. In vitro environments can consist of, but are not limitedto, processes or reactions that occur in a test tube. The term “in vivo”refers to the natural environment and to processes or reactions thatoccur within a natural environment (e.g., in an animal or a cell).

DESCRIPTION OF THE INVENTION

The description below provides exemplary embodiments of the presentinvention. It should be understood that the invention is not limited tothese exemplary embodiments.

Utility of the Invention

Exemplary uses of the invention are provided below. The presentinvention is not limited to these particular exemplary uses.

In some embodiments, the present invention provides a method comprising:providing an uncapped RNA comprising primary RNA transcripts or RNAhaving a 5′-diphosphate, a capping enzyme system, and a modified capnucleotide; and contacting the uncapped RNA with the capping enzymesystem and the modified cap nucleotide under conditions whereinmodified-nucleotide-capped RNA is synthesized. In other embodiments, theinvention provides a kit comprising a capping enzyme system and amodified cap nucleotide.

The invention finds use in research, including clinical research, aswell as in commercial and therapeutic applications, particularly withrespect to in vitro and in vivo production of RNA and polypeptides, andfor capture or isolation of uncapped RNA comprising primary RNAtranscripts or RNA having a 5′-diphosphate in a mixed population thatalso includes other RNA molecules.

During development of embodiments of the present invention, it wasdetermined that vaccinia virus capping enzyme or a poxvirus cappingenzyme that has similar enzymatic activities can utilize modified capnucleotides as substrates to cap uncapped RNA comprising primary RNAtranscripts or RNA having a 5′-diphosphate. Thus, in some embodiments,the present invention provides a method for catalyzing formation of amodified-nucleotide-capped RNA comprising the step of contacting an RNAtranscript with a capping enzyme system, such as poxvirus cappingenzyme, and modified cap nucleotide under conditions permissive to theformation of the modified-nucleotide-capped RNA. In some embodiments ofthe method, the uncapped RNA comprising primary RNA transcripts or RNAhaving a 5′-diphosphate is in a biological sample. In some embodiments,the uncapped RNA is mRNA in the biological sample. In some embodiments,the uncapped RNA in the biological sample comprises small primary RNAtranscripts that are not mRNA. In some embodiments, the uncapped RNA inthe biological sample comprises small nuclear RNA (snRNA), micro RNA(miRNA), or another primary RNA transcript. In some embodiments, theuncapped RNA comprising primary RNA transcripts or RNA having a5′-diphosphate is synthesized by an RNA polymerase in an in vitrotranscription reaction or RNA amplification reaction, or by a replicasein an in vitro replication reaction. In some cases, up to essentially100% of the uncapped RNA comprising primary RNA transcripts or RNAhaving a 5′-diphosphate in a population of RNA molecules is capped(e.g., see Examples). Thus, the present invention provides compositions,kits and methods for significantly improving synthesis of capped RNAtranscripts (e.g., mRNA). The present invention provides a method, andkits for performing the method, which use a capping enzyme, such asvaccinia capping enzyme or poxvirus capping enzyme, and a modified capnucleotide to synthesize modified-nucleotide-capped RNA with higherefficiency (e.g., with a capping efficiency approaching 100%) and inhigher yields than is obtained by co-transcriptional capping using adinucleotide cap analog. In some embodiments, the capping efficiency isgreater than 80%. In some embodiments, the capping efficiency is greaterthan 85%. In some embodiments, capping efficiency is greater than 90%.In some embodiments, capping efficiency is greater than 97%. In someembodiments, capping efficiency is greater than of 99%. Thus, thepresent invention provides compositions, kits and methods forsignificantly improving synthesis of capped RNA transcripts (e.g.,mRNA).

The methods and kits of embodiments of the invention result in amodified-nucleotide-capped RNA with the modified cap nucleotide in thecorrect orientation. In some embodiments, the method yields novelmodified-nucleotide-capped RNA compositions having a cap 0 structurethat have improved translational efficiency in vitro and/or in vivocompared to RNA compositions having the same sequence but which areuncapped or which are capped with an unmodified m⁷G cap nucleotide. Insome embodiments, the method yields novel modified-nucleotide-capped RNAcompositions having a cap I structure, which further improvestranslational efficiency. In some embodiments, the modifiednucleotide-capped RNA has a 3′ poly(A) tail. In still other embodiments,the method of the invention is used to selectively label uncapped RNAcomprising a primary RNA transcript or RNA having a 5′-diphosphate withan affinity tag (e.g., biotin), or with a fluorescent dye or otherdetectable molecule, thereby permitting selective capture, isolation,detection, quantification and/or assay of the modified-nucleotide-cappedRNA molecules in a population of other molecules. In some embodiments ofthe invention, the method uses the modified-nucleotide-capped RNA, in apopulation that also comprises other RNA molecules, to selectivelycapture or isolate the modified-nucleotide-capped RNA without capturingor isolating the RNA molecules that do not comprise a modified capnucleotide. For example, but without limitation, methods of theinvention can be used to isolate prokaryotic mRNA in a mixture ofprokaryotic total RNA or in a mixture of prokaryotic and eukaryotictotal RNA, such as from a eukaryotic cell infected by a prokaryoticpathogen (e.g., Mycobacterium) or from a eukaryotic cell in symbiosiswith a prokaryote (e.g., Rhizobium in a legume root nodule). Theinvention further provides kits that use the novel methods of theinvention for obtaining modified-nucleotide-capped RNA molecules,including novel compositions of such molecules that have improved invivo and/or in vitro translational efficiencies or other properties thatare useful for other applications described herein. Thus, the inventionprovides novel methods, kits and compositions with significant benefitsand advantages over the prior art for a variety of research, commercial,and therapeutic applications.

Methods and Kits for Capping Uncapped RNA

Embodiments of the present invention provide methods and kits forobtaining a modified-nucleotide-capped RNA and compositions comprisingmodified-nucleotide-capped RNA molecules not previously known in theart.

In one embodiment, the invention is a kit comprising a capping enzymesystem and a modified cap nucleotide. A method of the invention forobtaining modified-nucleotide-capped RNA comprises: (a) providing anuncapped RNA comprising primary RNA transcripts or RNA having a5′-diphosphate, a capping enzyme system, and a modified cap nucleotide;and contacting the uncapped RNA with the capping enzyme system and themodified cap nucleotide under conditions whereinmodified-nucleotide-capped RNA is synthesized.

In some embodiments of the invention, the kit further comprisesS-adenosyl-methionine or, in some embodiments, S-adenosyl-ethionine, inview of the finding that 7-ethylguanosine-containing capped RNAsynthesized in vitro by reovirus capping enzyme in the presence ofS-adenosyl-ethionine was equally active in translation and binding toribosomes as 7-methylated capped RNA (Furuichi, Y et al., J. Biol.Chem., 254: 6732-6738, 1979). In some embodiments of the method, themethod further comprises providing S-adenosyl-methionine orS-adenosyl-ethionine. In some embodiments of the method, the cappingenzyme system comprises guanine-7-methyltransferase activity and theuncapped RNA is contacted with the capping enzyme system and themodified cap nucleotide is synthesized in the presence ofS-adenosyl-methionine or S-adenosyl-ethionine, whereby themodified-nucleotide-capped RNA synthesized has a methyl group or anethyl group, respectively, on the N7 position of the guanine in themodified cap nucleotide. In some embodiments of the method, themodified-nucleotide-capped RNA is synthesized in the absence ofS-adenosyl-methionine or S-adenosyl-ethionine, wherein the modified capnucleotide does not have a methyl group on the nitrogen at the 7position of the guanine.

In some embodiments of the invention, the kit comprising a cappingenzyme system, a modified cap nucleotide, and S-adenosyl-methioninefurther comprises an enzyme having mRNA (nucleoside-2′-O—)methyltransferase activity. In some embodiments of the invention, themethod of contacting the uncapped RNA transcript with the capping enzymesystem and the modified cap nucleotide under conditions whereinmodified-nucleotide-capped RNA is synthesized further comprises the stepof contacting the modified-nucleotide-capped RNA with an mRNA(nucleoside-2′-O—) methyltransferase and S-adenosyl-methionine underconditions wherein a modified-nucleotide-capped RNA having a cap Istructure (i.e., having a methylated 2′-hydroxyl group in thepenultimate nucleotide at the 5-end of the RNA) is obtained. In someembodiments, the step of contacting the primary RNA transcript with thecapping enzyme system and the modified cap nucleotide under conditionswherein modified-nucleotide-capped RNA is synthesized and the step ofcontacting the modified-nucleotide-capped RNA with an mRNA(nucleoside-2′-O—) methyltransferase and S-adenosyl-methionine arecarried out concurrently in the same reaction mixture. In someembodiments, the enzyme having mRNA (nucleoside-2′-O—) methyltransferaseactivity is encoded by poxvirus DNA. In some embodiments, the enzymehaving mRNA (nucleoside-2′-O—) methyltransferase activity is encoded byvaccinia virus DNA. In some embodiments, the mRNA (nucleoside-2′-O—)methyltransferase is purified from virions. In other embodiments, themRNA (nucleoside-2′-O—) methyltransferase is purified from a recombinantsource. In some embodiments, the mRNA (nucleoside-2′-O—)methyltransferase is purified from E. coli cells that express thepoxvirus gene that is cloned in a plasmid or other vector. In someembodiments, the mRNA (nucleoside-2′-O—) methyltransferase is purifiedfrom E. coli cells that express the vaccinia gene that is cloned in aplasmid or other vector. In some embodiments, the enzyme having mRNA(nucleoside-2′-O—) methyltransferase activity is encoded by a yeast orfungal gene. In other embodiments, the enzyme is encoded by a nematode,mammalian or other metazoan gene, whether from a wild-type orrecombinant source. The invention is not limited by the particularsource of the enzyme, so long as it is active in methylating the2′-position of the penultimate nucleotide at the 5-end ofmodified-nucleotide-capped RNA.

In some embodiments, the method further comprises the step of contactingthe modified-nucleotide-capped RNA with an mRNA (nucleoside-2′-O—)methyltransferase in the presence of S-adenosyl-methionine underconditions wherein a modified-nucleotide-capped RNA with a cap Istructure (i.e., having a methyl group on the 2′-hydroxyl of thepenultimate nucleotide at the 5-end) is obtained. In some embodiments ofthe method, the modified-nucleotide-capped RNA having a cap I structureis synthesized in the presence of the capping enzyme system and the mRNA(nucleoside-2′-O—) methyltransferase in the same reaction mixture. Insome embodiments, the enzyme having mRNA (nucleoside-2′-O—)methyltransferase activity is encoded by a poxvirus DNA. In someembodiments, the mRNA (nucleoside-2′-O—) methyltransferase is purifiedfrom poxvirus virions, whereas in other embodiments, the mRNA(nucleoside-2′-O—) methyltransferase is purified from a recombinantsource, such as, from E. coli cells which express the gene that encodesthe enzyme and which is cloned in a plasmid or other vector. In someembodiments, the enzyme having mRNA (nucleoside-2′-O—) methyltransferaseactivity is encoded by vaccinia virus DNA. In some embodiments, the mRNA(nucleoside-2′-O—) methyltransferase is purified from vaccinia virions,whereas in other embodiments, the mRNA (nucleoside-2′-O—)methyltransferase is purified from a recombinant source, such as from E.coli cells which express the vaccinia gene for the enzyme that is clonedin a plasmid or other vector.

During the course of the investigations related to the development ofembodiments of the present invention, we found that an enzyme with mRNA(nucleoside-2′-O—) methyltransferase activity can methylate the2′-hydroxyl group of the penultimate nucleotide at the 5-end of said5′-capped RNA, including modified-nucleotide-capped RNA, irrespective ofwhether the N⁷-position of the guanine base of the cap nucleotide,including a modified cap nucleotide, is methylated. Therefore, withrespect to the present invention, when we refer to “capped RNA having acap 0 structure”, including a “modified-nucleotide-capped RNA having acap 0 structure”, we mean the RNA that results from the addition of thecap nucleotide to the 5′-end of primary RNA by the capping enzymesystem, whether or not the cap nucleotide or the modified cap nucleotidehas a methyl group on the N⁷-position of the guanine base, and when werefer to “capped RNA having a cap I structure”, including a“modified-nucleotide-capped RNA having a cap I structure”, we mean the5′-capped RNA that has a methyl group on the 2′-hydroxyl group of thepenultimate nucleotide at the 5-end of said 5′-capped RNA, in mostcases, but not exclusively, by the action of an enzyme with mRNA(nucleoside-2′-O—) methyltransferase activity.

In some embodiments, the kits and methods for synthesizingmodified-nucleotide-capped RNA having a cap 0 or cap I structure, withor without a poly(A) tail, and the compositions comprising amodified-nucleotide-capped RNA having a cap 0 or cap I structure, withor without a poly(A) tail, are further used for in vitro or in vivotranslation of proteins or polypeptides. For example, some embodimentsof the invention comprise kits, methods and compositions, not previouslyknown in the art, that provide modified-nucleotide-capped RNAs that aretranslated into proteins with equal or higher efficiency in cells invivo and/or in cell-free extracts in vitro compared to unmodifiedm⁷G-capped RNA molecules having the same sequence. In some embodimentsin which translation efficiency is equal to unmodified m⁷G-capped RNA,the modified-nucleotide-capped RNA has benefits in particularapplications, cells, or conditions. The present inventors found thatsome compositions of modified-nucleotide capped RNA synthesized usingthe methods and kits of the invention were translated in vivo or invitro with higher efficiency than the same RNA molecules with anunmodified m⁷G-cap, whether the caps had a cap 0 or a cap I structure(e.g., see Examples). Thus, in some experiments,modified-nucleotide-capped RNA comprising an N⁷-Me-2′-amino-2′-dG-cappedRNA or an N⁷-Me-O⁶-Me-G-capped RNA with caps having either a cap 0 or acap I structure and poly(A) tails on their 3′-termini were translated invivo or in vitro with higher efficiency than the corresponding RNAmolecules with an unmodified m⁷G-cap. In other experiments wherein adifferent primary RNA transcript was used to synthesizemodified-nucleotide-capped RNA, a different spectrum of modified capnucleotides (e.g., O⁶-Me-GTP, 2′-F-2′dGTP, 2′-O-Me-GTP, and2′-azido-dGTP) resulted in synthesis of modified-nucleotide-capped RNAsthat exhibited higher levels of in vivo or in vitro translation (e.g.,in some cases, higher than with the corresponding RNA molecules with anunmodified m⁷G-cap) and the relative translation levels varied withdifferent types of cells or cell-free translation systems.

In other embodiments of the kits and method for synthesizing amodified-nucleotide-capped RNA for in vitro or in vivo translation ofproteins or polypeptides, the modified cap nucleotide is3′-O-methylguanosine-5′-triphosphate or2′-O-methylguanosine-5′-triphosphate, in which embodiments, themodified-nucleotide-capped RNA synthesized using the kit and methodshould be the same as the capped RNA obtained in an in vitrotranscription reaction using the respective m₂ ^(7, 3′-O) GpppG or m₂^(7, 2′-O) GpppG ARCA. The present invention is beneficial because suchmodified-nucleotide-capped RNA molecules having a modified capnucleoside with a 3′- or 2′-O-methyl group can be synthesized withhigher efficiency and in higher yields using a method or kit of theinvention than the molecules synthesized by in vitro transcriptionreaction using the respective ARCA that have been reported to betranslated in vitro and in vivo, with higher efficiency than unmodifiedm⁷G-capped RNA. In other embodiments in which the modified capnucleotide is 3′-deoxyguanosine-5′-triphosphate, themodified-nucleotide-capped RNA obtained using the kit or method of theinvention is identical to the capped RNA obtained by in vitrotranscription in the presence of the m⁷(3′)dGpppG ARCA, which has alsobeen reported to be translated in vitro with higher efficiency. Thus,the kits and methods of the invention are more desirable in terms ofyield, purity, cost and/or time compared to existing techniques forobtaining such capped RNA molecules using a dinucleotide ARCA in an invitro transcription reaction.

The present inventors also found that capped RNA molecules obtained byin vitro transcription of a DNA template in the presence of adinucleotide cap analog, such as but not limited to an m₂ ^(7, 3′-O)GpppG or m₂ ^(7, 2′-O) GpppG ARCA, can be 2′-O-methylated by mRNA(nucleoside-2′-O—) methyltransferase in an in vitro reaction containingS-adenosyl-methionine. Thus, one other embodiment of the presentinvention is a method for synthesizing a modified-nucleotide-capped RNAwith a cap I structure (i.e., having a methylated 2′-hydroxyl group inthe penultimate nucleotide at the 5-end), said method comprising:providing a capped RNA molecule having a cap 0 structure that wasobtained using a dinucleotide cap analog in an in vitro transcriptionreaction (e.g., m₂ ^(7, 3′-O) G-capped RNA or m₂ ^(7, 2′-O) G-cappedRNA); and contacting the modified-nucleotide-capped RNA having a cap 0structure with mRNA (nucleoside-2′-O—) methyltransferase andS-adenosyl-methionine under conditions wherein amodified-nucleotide-capped RNA with a cap I structure is synthesized.

The invention also provides kits and methods for obtainingmodified-nucleotide-capped RNA that has a poly(A) tail. This can bebeneficial because capped RNA that has a poly(A) tail is more stable andis translated with higher efficiency in vivo, and sometimes in vitro,than the same RNA that lacks a poly(A) tail. Thus, in some embodiments,the kit further comprises poly(A) polymerase. In some embodiments of theinvention, the method further comprises the step of contacting themodified-nucleotide-capped RNA, having either a cap 0 or a cap Istructure, with poly(A) polymerase and ATP under conditions whereinmodified-nucleotide-capped RNA having a poly(A) tail is synthesized. Insome embodiments, the length of the poly(A) tail synthesized is about 30nucleotides. In other embodiments, the length of the poly(A) tailsynthesized is about 30 nucleotides to at about 100 nucleotides. Inother embodiments, the length of the poly(A) tail synthesized is about100 nucleotides to at about 200 nucleotides. In still other embodiments,the length of the poly(A) tail synthesized is about 200 nucleotides toabout 400 nucleotides. In some embodiments, the length of the poly(A)tail synthesized is greater than 400 nucleotides. In some embodimentswherein the modified-nucleotide-capped RNA is translated in vitro incell-free extracts, the modified-nucleotide-capped RNA lacks a poly(A)tail. Without limitation, the poly(A) polymerase can be selected fromthe group consisting of Escherichia coli poly(A) polymerase and yeastpoly(A) polymerase. In some embodiments the poly(A) polymerase isrecombinant poly(A) polymerase encoded by the E. coli pcnB gene. Methodsfor polyadenylating RNA using a poly(A) polymerase are well known in theart and kits for such purpose are commercially available. For example,RNA can be polyadenylated using A-Plus™ poly(A) polymerase tailing kit(EPICENTRE Biotechnologies, Madison, Wis., USA) according to thedirections provided with the kit.

In some cases, an uncapped RNA is more difficult to cap if the sequenceof nucleotides at the 5′-terminus of the uncapped RNA to be capped iscomplementary to and anneals with another sequence that is within thesame RNA molecule or that is in a second RNA molecule present in thecapping enzyme reaction. For example, but without limitation, theuncapped RNA is more difficult or impossible to cap using a cappingenzyme system and a cap nucleotide if the sequence at the 5′-terminus ofthe uncapped RNA anneals to form a stable duplex, such as a hairpin,dimer or another duplex structure that involves at least the 5′-terminalnucleotide. The inventors found certain conditions for carrying out thecapping reaction using capping enzyme and a cap nucleotide wherein theefficiency of capping uncapped RNA that forms such a duplex involvingthe 5′-terminus is increased by at least 25 percent. Without being boundby theory, it is believed that the capping efficiency increases usingsuch conditions because the conditions disrupt or interfere withformation of the duplex involving the 5′-terminus, either by decreasingthe melting temperature (Tm) of the duplex, or by competitively bindingto nucleotides in close proximity to the 5′-terminus of the uncapped RNA(e.g., within about 3 to about 30 bases of the 5′-terminus of theuncapped RNA). Thus, one embodiment of the present invention is areaction mixture comprising a component that improves the efficiency ofcapping uncapped RNA that forms a duplex involving at least the5′-terminal nucleotide, said reaction mixture comprising a cappingenzyme system and a cap nucleotide, and additionally comprising at leastone of the following: (i) dimethylglycine (betaine) at, for example, afinal concentration of at least 0.5 M; (ii) a single-stranded bindingprotein at, for example, a concentration of at least 0.1 micrograms permicroliter; (iii) an RNA helicase at, for example, a concentration of atleast 0.1 micrograms per microliter; (iv) an RNA polymerase at, forexample, a concentration of at least 0.1 micrograms per microliter; or(v) a DNA polymerase at, for example, a concentration of at least 0.1micrograms per microliter, or functionally equivalent components. Oneembodiment is a kit comprising a capping enzyme system and a capnucleotide, and additionally comprising at least one of the following:(i) dimethylglycine (betaine); (ii) a single-stranded binding protein;(iii) an RNA helicase; (iv) an RNA polymerase; or (v) a DNA polymerase.One embodiment of a method comprises contacting the uncapped RNA withcapping enzyme system and the cap nucleotide in a reaction mixturecomprising at least one of the following: (i) dimethylglycine (betaine)at a final concentration of at least 0.5 M; (ii) a single-strandedbinding protein at a concentration of at least 0.1 micrograms permicroliter; (iii) an RNA helicase at a concentration of at least 0.1micrograms per microliter; (iv) an RNA polymerase at a concentration ofat least 0.1 micrograms per microliter; or (v) a DNA polymerase at aconcentration of at least 0.1 micrograms per microliter. The aboveembodiments of the reaction mixture, the kit, or the method are notlimited to use of a modified cap nucleotide. Any cap nucleotide that isa substrate for the capping enzyme system can be used. In some preferredembodiments, the cap nucleotide is a modified cap nucleotide. In somepreferred embodiments, the cap nucleotide is GTP. In embodiments of thereaction mixture, kit or method that comprise dimethylglycine (betaine),the compound is used as a zwitterion and is not used as a salt and thefinal concentration in the reaction mixture can be up to about 5 M. Insome embodiments of the reaction mixture, kit or method, thesingle-stranded binding protein is bacteriophage T4 gene 32 protein or aThermus single-stranded binding protein; however, the invention alsocontemplates use of another suitable single-stranded binding proteinthat binds to nucleotides in close proximity to the 5′-terminus of theuncapped RNA (e.g., within about 3 to about 30 bases of the 5′-terminusof the uncapped RNA). In some embodiments of the reaction mixture, kitor method, the RNA polymerase used is a T7-type RNA polymerase; in someembodiments, it is selected from the group consisting of T7 RNApolymerase, T3 RNA polymerase, and SP6 RNA polymerase. In embodiments ofthe reaction mixture, kit or method, that comprise DNA polymerase, theDNA polymerase is the large fragment of Bacillus DNA polymerase (i.e.,equivalent to the enzyme obtained by subtilisin digestion of the DNApolymerase holoenzyme or the clone thereof). In some embodiments of thereaction mixture, kit or method, the component is titrated in thereaction mixture to determine the optimal concentration for cappingusing the capping enzyme system, the cap nucleotide, and the particularuncapped RNA. In some embodiments, the method additionally comprises thestep of heating the uncapped RNA in water at a temperature of at least10 degrees above the melting temperature (i.e., Tm) of the duplex for atleast two minutes and then cooling rapidly on ice, prior to using it inthe capping reaction.

Types and Sources of Uncapped RNA

Exemplary types and sources of uncapped RNA useful in the methods andcompositions of embodiments of the invention are described below.

In some embodiments, the uncapped RNA is a primary RNA transcript,meaning an RNA having a 5′-triphosphate group from an in vivo or an invitro source, and the method comprises contacting the primary RNAtranscript with the capping enzyme system and the modified capnucleotide under conditions wherein modified-nucleotide-capped RNA issynthesized. For example, but without limitation, in some embodiments,the primary RNA transcript is uncapped mRNA from a prokaryotic source orthe primary RNA transcript is synthesized by in vitro or in vivotranscription of a DNA template using an RNA polymerase, or by in vitroor in vivo replication of an RNA template using a replicase (e.g.,Q-beta replicase). In some embodiments, the primary RNA transcript issynthesized in an in vitro transcription reaction using a T7-type RNApolymerase. Without limitation, in some embodiments, the T7-type RNApolymerases that is used to synthesize the primary RNA transcript isselected from the group consisting of T7 RNA polymerase, T3 RNApolymerase and SP6 RNA polymerase. In some embodiments, the primary RNAtranscript is obtained from an in vitro transcription reaction that ispart of an RNA amplification reaction. In some preferred embodiments,the RNA amplification reaction results in synthesis of sense RNA. Insome embodiments, the RNA amplification reaction results in synthesis ofanti-sense RNA. The primary RNA transcript can also be pre-miRNA orpre-snRNA from an in vivo source, or any other primary RNA transcripthaving a 5′-triphosphate from an in vivo or in vitro source.

In some embodiments, the uncapped RNA is RNA having a 5′-diphosphategroup rather than a primary RNA transcript having a 5′-triphosphategroup, and the method comprises contacting the RNA having a5′-diphosphate group with the capping enzyme system and the modified capnucleotide under conditions wherein modified-nucleotide-capped RNA issynthesized. In some embodiments in which uncapped RNA that has a5′-diphosphate group is used rather than a primary RNA transcript havinga 5′-triphosphate group, the capping enzyme system is a capping enzymesystem having RNA triphosphatase activity, even though such enzymaticactivity is not required. In other embodiments in which the RNA has a5′-diphosphate group is used, the method comprises use of a cappingenzyme system that lacks RNA triphosphatase activity. Thus, whenever amethod of the present invention described herein uses a primary RNAtranscript to synthesize a modified-nucleotide-capped RNA, it should beunderstood that the invention also includes the same method in which anRNA having a 5′-diphosphate group is used in place of a primary RNAtranscript having a 5′-triphosphate group. In some embodiments, the RNAthat has a 5′-diphosphate group is from an in vivo or an in vitrosource. The invention is not limited with respect to the source of theRNA that has a 5′-diphosphate. For example, but without limitation, insome embodiments, the RNA that has a 5′-diphosphate is obtained by usinga nucleoside-5′-diphosphate (“NDP”) to prime in vitro transcription ofDNA templates using an RNA polymerase. For example, in one embodiment,the RNA that has a 5′-diphosphate is synthesized using a T7-type RNApolymerase, such as T7, T3 or SP6 RNA polymerase, in an in vitrotranscription reaction, except that, instead of using only the fourribonucleoside-5′-triphosphates (i.e., collectively, “NTPs”, orindividually, “ATP”, “CTP”, “GTP” and “UTP”, each of which isgenerically referred to as an “NTP”) at appropriate concentrations knownin the art for such in vitro transcription (e.g., see Method 1), the NTPcorresponding to the first-transcribed or initiating nucleotide of theRNA in the reaction is replaced by a mixture of the NDP (i.e., the“nucleoside-5′-diphosphate”) and the NTP corresponding to thefirst-transcribed nucleotide. The ratio of NDP to NTP is set at betweenabout 4-to-1 to about 10-to-1, or, in other embodiments, even to about20-to-1, or more, in the in vitro transcription reaction mixture so thatthere is a higher probability that the RNA polymerase initiatestranscription by extension of the 3′-OH of the NDP, rather than byextension of the 3′-OH of the NTP. In other embodiments, the RNA thathas a 5′-diphosphate is obtained by contacting a primary RNA transcripthaving a 5′-triphosphate with an RNA triphosphatase under suitablereaction conditions. In other embodiments, the RNA having a5′-diphosphate is obtained by contacting an RNA having a 5′-cap with adecapping enzyme (e.g., Saccharomyces cerevisiae or human decappingenzyme) under suitable reaction conditions. In other embodiments, theRNA that has a 5′-diphosphate is obtained from an in vivo source, i.e.,from a biological sample.

In some embodiments, the uncapped RNA comprising primary RNA transcriptsor RNA having a 5′-diphosphate group for synthesizing amodified-nucleotide-capped RNA lacks a poly(A) tail on the 3′-terminus,such as is the case for most mRNA molecules from a prokaryotic bacterialsource. In some embodiments, the uncapped RNA comprising a primary RNAtranscript or RNA that has a 5′-diphosphate group has a poly(A) tail onthe 3′-terminus, which uncapped RNA is obtained from an in vivo source,such as an uncapped primary RNA transcript from a eukaryotic cell, or insome cases, which uncapped RNA is obtained from a prokaryotic cell, orfrom an in vitro source. In some embodiments the primary RNA transcriptwith the poly(A) tail is obtained by in vitro transcription of a DNAtemplate that encodes the poly(A) tail. In other embodiments, theprimary RNA transcript with the poly(A) tail is obtained by in vitropolyadenylation of a primary RNA transcript without a poly(A) tail usingATP and poly(A) polymerase. Thus, in some embodiments, the kit of theinvention further comprises poly(A) polymerase. In some embodimentswherein the modified-nucleotide-capped RNA that is synthesized using amethod of the invention lacks a poly(A) tail, the method furthercomprises the step of contacting the modified-nucleotide-capped RNA withpoly(A) polymerase and ATP under conditions wherein amodified-nucleotide-capped RNA having a poly(A) tail is synthesized.

In some embodiments of the invention, the uncapped RNA comprisingprimary RNA transcripts or RNA having a 5′-diphosphate that is used tosynthesize modified-nucleotide-capped RNA is from an in vitrotranscription reaction or an RNA amplification reaction that producessense RNA (e.g., but without limitation, by amplification of RNA fromone or a small number of cancer cells from a patient). In someembodiments, the RNA amplification reaction used to synthesize the senseRNA comprises the terminal tagging method described in U.S. PatentApplication No. 20050153333 of Sooknanan. In preferred embodiments ofthe invention, the uncapped RNA comprising primary RNA transcripts orRNA having a 5′-diphosphate that is used to synthesizemodified-nucleotide-capped RNA is prepared from a biological source bysubtractive hybridization, digestion and RNA amplification, whereby theuncapped RNA comprising primary RNA transcripts or RNA having a5′-diphosphate obtained is “condition-specific”, as defined elsewhereherein. In some preferred embodiments, the condition-specific RNA isobtained using the “cap-dependent subtraction” (“CDS”) method of thepresent invention, as described herein. In preferred embodiments, thecondition-specific uncapped RNA obtained using the CDS method is furtheramplified using a sense RNA amplification reaction. Thus, in someembodiments, the uncapped RNA comprising primary RNA transcripts or RNAhaving a 5′-diphosphate is condition-specific, and the method comprisescontacting the condition-specific uncapped RNA comprising primary RNAtranscripts or RNA having a 5′-diphosphate with the capping enzymesystem and the modified cap nucleotide under conditions whereincondition-specific modified-nucleotide-capped RNA is synthesized. Indifferent embodiments, the condition-specific modified-nucleotide-cappedRNA that is sythesized is selected from the group consisting of: cap 0modified-nucleotide-capped RNA with a poly(A) tail; cap Imodified-nucleotide-capped RNA with a poly(A) tail; cap 0modified-nucleotide-capped RNA lacking a poly(A) tail; and cap Imodified-nucleotide-capped RNA lacking a poly(A) tail.

Capping Enzyme System

Embodiments of the present invention utilize a capping enzyme system inmethods to synthesize modified-nucleotide-capped RNA, and in kits thatalso contain a modified cap nucleotide for use in the method. Thepresent invention is not limited by the type of capping enzyme utilized.Indeed, a variety of capping enzymes can be used in the presentinvention. In some embodiments, the capping enzyme system is purifiedfrom cells that express the capping enzymes from genes cloned in one ormore recombinant vectors. In some embodiments, the capping enzyme systemis purified from a natural source. In some embodiments, the cappingenzyme system is a poxvirus capping enzyme system. In some preferredembodiments, the capping enzyme system is vaccinia virus capping enzyme.In some embodiments, the vaccinia virus capping enzyme system ispurified from vaccinia virions. In other embodiments, the vaccinia viruscapping enzyme system is purified from a recombinant source. In apreferred embodiment, the capping enzyme system used in the method or ina kit of the invention comprises RNA guanyltransferase activity. In onepreferred embodiment, the capping enzyme system comprises RNAtriphosphatase and RNA guanyltransferase activity. In some preferredembodiments, the capping enzyme system comprises purified RNAguanyltransferase and guanine-7-methyltransferase activities, whichactivities can be from a native or recombinant source. In some preferredembodiments, the capping enzyme system comprises purified RNAtriphosphatase, RNA guanyltransferase, and guanine-7-methyltransferaseactivities, which activities can be from a native or recombinant source.The capping enzyme system can comprise only one protein having all ofthese enzyme activities. One such preferred embodiment comprises apoxvirus capping enzyme system. One such preferred embodiment comprisesa vaccinia capping enzyme system. Alternatively, the capping enzymesystem can comprise two or three different proteins or polypeptideshaving RNA triphosphatase, RNA guanyltransferase, andguanine-7-methyltransferase activity. Some embodiments of the inventioncomprise kits or methods, wherein the capping enzyme system used in thekit or method comprises two or three enzymes having RNA triphosphatase,RNA guanyltransferase, and guanine-7-methyltransferase activity, eachenzyme can be from the same source or they can be from two or threedifferent sources. For example, but without limitation, in someembodiments, the kits or methods provide a capping enzyme system whereinthe polypeptide having RNA triphosphatase activity and/or thepolypeptide having guanine-7-methyltransferase activity are from asingle source (e.g., from Saccharomyces cerevisiae) and the polypeptidehaving RNA guanyltransferase activity is from another source (e.g., apolypeptide encoded by a poxvirus gene); and in other embodiments, thekits or methods provide a capping enzyme system wherein the polypeptideshaving each of the three enzymatic activities is from a differentnatural or recombinant source. If the capping enzyme system comprisestwo or three different enzymes or polypeptides, the enzymes can be usedsequentially or, more preferably, they can be used together in a singlereaction mixture. In some embodiments, the capping enzyme system hasguanine 7-methyltransferase activity and the method comprises contactingthe uncapped RNA with the capping enzyme system in the presence ofS-adenosyl-methionine or S-adenosyl-ethionine under conditions whereinthe capping enzyme system, including the guanine-7-methyltransferaseactivity of said capping enzyme system, is active andmodified-nucleotide-capped RNA having, respectively, a methyl or anethyl group on the nitrogen at the 7 position of guanine is synthesized.In some embodiments, the enzyme that has guanine-7-methyltransferaseactivity is poxvirus guanine-7-methyltransferase. In some embodiments,the enzyme having guanine-7-methyltransferase activity is a component ofthe vaccinia capping enzyme.

Modified Cap Nucleotides

Exemplary modified cap nucleotides of embodiments of the presentinvention are described below.

In some preferred embodiments, the modified cap nucleotide used in themethod or kit of the invention comprises a modified 2′- or3′-deoxyguanosine-5′-triphosphate (also called guanine 2′- or3′-deoxyribonucleoside-5′-triphosphate) wherein the 2′- or 3′-deoxyposition of the sugar moiety is substituted with a moiety comprising anamino group, an azido group, a fluorine group, a methoxy group, a thiol(or mercapto) group, or a methylthio (or methylmercapto) group, orwherein the O6 oxygen of the guanine base is replaced by a thiol (ormercapto) group or by a methylthio (or methylmercapto) group. In otherpreferred embodiments, the modified cap nucleotide comprises modifiedguanosine-5′-triphosphate (also called guanineribonucleoside-5′-triphosphate) wherein the N1 nitrogen of the guaninebase is modified by being substituted with a methyl group, or whereinthe O6 oxygen of the guanine base is modified by being substituted withan alkyl group, or wherein the O6 oxygen of the guanine base is replacedby a thiol (or mercapto) group or by a methylthio (or methylmercapto)group. In one preferred embodiment, the alkyl group on the O6 oxygen ofthe guanine base of the modified guanosine-5′-triphosphate is a methylgroup. In other embodiments of the method or the kit, the modified capnucleotide is 3′-deoxyguanosine-5′-triphosphate.

In some embodiments, the kit or method comprises a capping enzyme systemand a modified cap nucleotide, wherein the modified cap nucleotidecomprises a modified guanine nucleoside-5′-triphosphate selected fromthe group consisting of: 2′-O-methylguanosine-5′-triphosphate (orguanine 2′-O-methyl-ribonucleoside-5′-triphosphate, or 2′-O-methyl-GTP,or 2′-O-Me-GTP); 3′-O-methylguanosine-5′-triphosphate (or guanine3′-O-methyl-ribonucleoside-5′-triphosphate, or 3′-O-methyl-GTP or3′-O-Me-GTP); 2′-amino-2′-deoxyguanosine-5′-triphosphate (or guanine2′-amino-2′-deoxyribonucleoside-5′-triphosphate, or 2′-NH₂-2′-dGTP or2′-amino-2′-dGTP); 3′-amino-3′-deoxyguanosine-5′-triphosphate (orguanine 3′-amino-3′-deoxyribonucleoside-5′-triphosphate, or3′-NH₂-3′-dGTP or 3′-amino-3′-dGTP);2′-azido-2′-deoxyguanosine-5′-triphosphate (or guanine2′-azido-2′-deoxyribonucleoside-5′-triphosphate, or 2′-N₃-dGTP, or2′-azido-2′-dGTP); 3′-azido-3′-deoxyguanosine-5′-triphosphate (orguanine 3′-azido-3′-deoxyribonucleoside-5′-triphosphate, or 3′-N₃-dGTP,or 3′-azido-3′-dGTP); 2′-fluoro-2′-deoxyguanosine-5′-triphosphate (orguanine 2′-fluoro-2′-deoxyribonucleoside-5′-triphosphate, or2′-fluoro-2′-dGTP, or 2′-F-2′-dGTP);3′-fluoro-3′-deoxyguanosine-5′-triphosphate (or guanine3′-fluoro-3′-deoxyribonucleoside-5′-triphosphate, or 3′-fluoro-3′-dGTPor 3′-F-3′-dGTP); 2′-mercapto-2′-deoxyguanosine-5′-triphosphate (orguanine 2′-mercapto-2′-deoxyribonucleoside-5′-triphosphate or2′-mercapto-2′-dGTP or 2′-SH-2′-dGTP);3′-mercapto-3′-deoxyguanosine-5′-triphosphate (or guanine3′-mercapto-3′-deoxyribonucleoside-5′-triphosphate or3′-mercapto-3′-dGTP or 3′-SH-3′-dGTP);2′-amino-2′,3′-dideoxyguanosine-5′-triphosphate (orguanine-2′-amino-2′,3′-dideoxyribonucleic acid-5′-triphosphate, or2′-amino-2′,3′-ddGTP); 3′-amino-2′,3′-dideoxyguanosine-5′-triphosphate(or guanine-3′-amino-2′,3′-dideoxyribonucleic acid-5′-triphosphate, or3′-amino-2′,3′-ddGTP); 2′-azido-2′,3′-dideoxyguanosine-5′-triphosphate(or guanine-2′-azido-2′,3′-dideoxyribonucleic acid-5′-triphosphate, or2′-azido-2′,3′-ddGTP); 3′-azido-2′,3′-dideoxyguanosine-5′-triphosphate(or guanine-3′-azido-2′,3′-dideoxyribonucleic acid-5′-triphosphate, or3′-azido-2′,3′-ddGTP);2′-mercapto-2′,3′-dideoxyguanosine-5′-triphosphate (orguanine-2′-mercapto-2′,3′-dideoxyribonucleic acid-5′-triphosphate, or2′-mercapto-2′,3′-ddGTP, or 2′-SH-2′,3′-ddGTP); and3′-mercapto-2′,3′-dideoxyguanosine-5′-triphosphate (orguanine-3′-mercapto-2′,3′-dideoxyribonucleic acid-5′-triphosphate, or3′-mercapto-2′,3′-ddGTP or 3′-SH-2′,3′-ddGTP).

In other embodiments, wherein the modified cap nucleotide comprises amodified guanine nucleoside-5′-triphosphate in which the guanine base ismodified, the modified cap nucleotide is selected from the groupconsisting of: N¹-methylguanosine-5′-triphosphate (or N¹-methylguanineribonucleoside-5′-triphosphate or N¹-methyl-GTP or N¹-Me-GTP);O⁶-methylguanosine-5′-triphosphate (or O⁶-methylguanineribonucleoside-5′-triphosphate or O⁶-methyl-GTP or O⁶-Me-GTP);6-thioguanosine-5′-triphosphate (or6-mercapto-guanosine-5′-triphosphate, or 6-thioguanineribonucleoside-5′-triphosphate, or 6-mercaptoguanineribonucleoside-5′-triphosphate, or 6-thio-GTP, or 6-SH-GTP);6-methylthioguanosine-5′-triphosphate (or6-methylmercapto-guanosine-5′-triphosphate, or 6-methylthioguanineribonucleoside-5′-triphosphate, or 6-methylmercaptoguanineribonucleoside-5′-triphosphate, or 6-methylthio-GTP, or6-methylmercapto-GTP, or 6-CH₃S-GTP);6-thio-2′-deoxyguanosine-5′-triphosphate (or6-thioguanine-2′-deoxyribonucleic acid-5′-triphosphate, or6-thio-2′-dGTP); 6-thio-3′-deoxyguanosine-5′-triphosphate (or6-thioguanine-3′-deoxyribonucleic acid-5′-triphosphate, or6-thio-3′-dGTP); 6-methylthio-2′-deoxyguanosine-5′-triphosphate (or6-methylthioguanine-2′-deoxyribonucleic acid-5′-triphosphate, or6-methylthio-2′-dGTP); and6-methylthio-3′-deoxyguanosine-5′-triphosphate (or6-methylthioguanine-3′-deoxyribonucleic acid-5′-triphosphate, or6-methylthio-3′-dGTP).

In still other embodiments of the kit or the method, the modified capnucleotide is 3′-deoxyguanosine-5′-triphosphate (or guanine3′-deoxyribonucleoside-5′-triphosphate, or 3′-dGTP), but not2′-deoxyguanosine-5′-triphosphate (or guanine2′-deoxyribonucleoside-5′-triphosphate, or dGTP or 2′-dGTP).

The invention is not limited to use of the modified cap nucleotidescomprising a modified sugar moiety or a modified base described above.The modified cap nucleotide that is used for the method can comprise anymodified guanine nucleoside-5′-triphosphate that is compatible with theenzymatic activities of the capping enzyme systems used in the method.

Modified-Nucleotide-Capped RNA Compositions

The present invention also comprises compositions comprising themodified-nucleotide-capped RNA made using kits and methods of thepresent invention. For example, composition are provided that are madeusing a kit comprising a capping enzyme and a modified cap nucleotide ora composition made using the method comprising contacting an uncappedRNA comprising a primary RNA transcript or an RNA having a5′-diphosphate with a modified cap nucleotide and a capping enzymesystem, wherein a modified-nucleotide-capped RNA is synthesized. Thus,the present invention includes new compositions ofmodified-nucleotide-capped RNA not previously known in the art.

Without limitation, the invention comprises a composition of amodified-nucleotide-capped RNA synthesized using a capping enzyme systemand a modified cap nucleotide, wherein said modified-nucleotide-cappedRNA is synthesized using a modified cap nucleotide comprising a modified2′- and/or 3′-deoxyguanosine-5′-triphosphate: wherein the respective 2′-or 3′-deoxy position of the sugar moiety is substituted with a moietycomprising an amino group, an azido group, a fluorine group, a thiol (ormercapto) group or a methylthio (or methylmercapto) group, or whereinthe O6-oxygen of the guanine base is replaced by a thiol group or by amethylthio group. In other preferred embodiments, the inventioncomprises a composition of a modified-nucleotide-capped RNA synthesizedusing a capping enzyme system and a modified cap nucleotide, whereinsaid modified-nucleotide-capped RNA is synthesized using a modified capnucleotide comprising a modified guanosine-5′-triphosphate wherein theN1 nitrogen of the guanine base is modified by being substituted with amethyl group or the O6 oxygen of the guanine base is modified by beingsubstituted with an alkyl group (e.g., a methyl group) or the O6 oxygenof the guanine base is replaced by a thiol group or by a methylthiogroup. In still other embodiments, the invention comprises any of theabove compositions of a modified-nucleotide-capped RNA, wherein themodified-nucleotide-capped RNA composition additionally comprises a2′-O-methyl group on the 5′-penultimate nucleotide, a poly(A) tail onthe 3′-terminus, or both a 2′-O-methyl group on the 5′-penultimatenucleotide and a poly(A) tail on the 3′-terminus. The invention alsocomprises compositions of modified-nucleotide-capped RNA that have a2′-O-methyl group on the 5′-penultimate nucleotide or both a 2′-O-methylgroup on the 5′-penultimate nucleotide and a poly(A) tail on the3′-terminus wherein said modified-nucleotide-capped RNA is synthesizedusing a modified cap nucleotide selected from among: (i)2′-deoxyguanosine-5′-triphosphate; (ii)3′-deoxyguanosine-5′-triphosphate; and (iii) 2′- or3′-deoxyguanosine-5′-triphosphate wherein the respective 2′- or 3′-deoxyposition is substituted with a methoxy group.

In still other embodiments, the composition comprises amodified-nucleotide-capped RNA wherein the modified cap nucleotide hasan amino or an azido group on the 2′- or 3′-position of the sugar or athiol group in place of the O6-oxygen of the guanine base and saidamino, azido, or thiol group is chemically joined to an affinity tagmolecule. In other embodiments, the composition ofmodified-nucleotide-capped RNA that comprises the affinity tagadditionally comprises: (i) a 2′-O-methyl group on the 5′-penultimatenucleotide; (ii) a poly(A) tail on the 3′-terminus; or (iii) both a2′-O-methyl group on the 5′-penultimate nucleotide and a poly(A) tail onthe 3′-terminus. In some preferred embodiments, wherein themodified-nucleotide-capped RNA comprises an affinity tag, thecomposition additionally comprises an affinity-tag-binding molecule thatis bound to the affinity tag. Still further, the invention alsocomprises embodiments of the modified-nucleotide-capped RNA that isbound through the affinity tag to an affinity-tag-binding molecule,whether said affinity-tag-binding molecule is free or attached to asurface. In some preferred embodiments of any of the above compositionscomprising an affinity tag molecule, the affinity tag molecule comprisesbiotin. In some embodiments of any of the above compositions wherein thecomposition additionally comprises an affinity-tag-binding molecule thatis bound to the affinity tag, the affinity-tag-binding molecule isavidin or streptavidin.

In still other embodiments, the composition comprises amodified-nucleotide-capped RNA wherein the modified cap nucleotide hasan amino or an azido group on the 2′- or 3′-position of the sugar or athiol group in place of the O6-oxygen of the guanine base and saidamino, azido, or thiol group is chemically joined to a detectable dye(e.g., a fluorescein dye, an alexa dye, a Cy dye, or another dye knownin the art) or to a detectable protein, such as, but not limited to, aphycobiliprotein. In other embodiments, the composition ofmodified-nucleotide-capped RNA that comprises the detectable dyemolecule additionally comprises: (i) a 2′-O-methyl group on the5′-penultimate nucleotide; (ii) a poly(A) tail on the 3′-terminus; or(iii) both a 2′-O-methyl group on the 5′-penultimate nucleotide and apoly(A) tail on the 3′-terminus. In different embodiments of any of theabove compositions, the detectable dye or detectable protein cancomprise any detectable moiety for such purpose that is known in theart, including, but not limited to, a detectable dyes described in“Handbook of Fluorescent Probes and Research Products”, Ninth Edition,by R. P. Hoagland, Molecular Probes, Inc.

The compositions comprising a modified-nucleotide-capped RNA can be usedfor a variety of applications. For example, but without limitation, amodified-nucleotide-capped RNA can be used as a substrate for in vivotranslation, except in cases such as that of amodified-nucleotide-capped RNA comprising an N¹-methylguanosine modifiedcap nucleotide, which is translated poorly or not at all. Therefore, amodified-nucleotide-capped RNA having N¹-methylguanosine in the modifiedcap nucleotide provides a negative control for studies involving invitro or in vivo translation. Thus, the invention also includes acomposition comprising a modified-nucleotide-capped RNA having anN¹-methylguanosine in the modified cap nucleotide.

The inventors found that modified-nucleotide-capped RNA comprising theN¹-methylguanosine modified cap nucleoside is not methylated by theguanine-7-methyltransferase activity of the capping enzyme system.However, in general, modified-nucleotide-capped RNA comprising othermodified cap nucleotides are N7-methylated by the capping enzyme systemin the presence of S-adenosyl-methionine. Thus, the invention comprisescompositions of modified-nucleotide-capped RNA comprising an N7-methylgroup on the modified cap nucleotide, as well as compositions ofmodified-nucleotide-capped RNA that lack the N7-methyl group (e.g., ifthe synthesis is carried out in the absence of S-adenosyl-methionine).In some embodiments, the capping reaction is carried out in the presenceof S-adenosyl-methionine, in which embodiments, the invention comprisescompositions of modified-nucleotide-capped RNA comprising an N7-ethylgroup on the modified cap nucleotide. Thus, by way of example, but notof limitation, in some embodiments, the invention comprises novelcompositions of a modified-nucleotide-capped RNA with any of thefollowing modified cap nucleosides: N¹-methylguanosine;O⁶-methylguanosine; N⁷-methyl-O⁶-methylguanosine (orN⁷,O⁶-dimethylguanosine); 2′-O-methylguanosine; 3′-O-methylguanosine;2′-amino-2′-deoxyguanosine; N⁷-methyl-2′-amino-2′-deoxyguanosine;3′-amino-3′-deoxyguanosine; N⁷-methyl-3′-amino-3′-deoxyguanosine;2′-azido-2′-deoxyguanosine; N⁷-methyl-2′-azido-2′-deoxyguanosine;3′-azido-3′-deoxyguanosine; N⁷-methyl-3′-azido-3′-deoxyguanosine;2′-fluoro-2′-deoxyguanosine; N⁷-methyl-2′-fluoro-2′-deoxyguanosine;3′-fluoro-3′-deoxyguanosine; N⁷-methyl-3′-fluoro-3′-deoxyguanosine; and3′-deoxyguanosine. In other embodiments, the invention comprises amodified-nucleotide-capped RNA composition, including any of thecompositions listed above, that additionally has a methyl group on the2′-hydroxyl group of the penultimate ribonucleotide at the 5-end (i.e.,has a cap I structure). The present invention further comprises a novelcomposition of a modified-nucleotide-capped RNA having a cap Istructure, wherein the modified-nucleotide-capped RNA having a cap Istructure has a modified cap nucleoside selected from the groupconsisting of: N⁷-methyl-2′-O-methylguanosine;N⁷-methyl-3′-O-methylguanosine; N⁷-methyl-2′-deoxyguanosine; andN⁷-methyl-3′-deoxyguanosine, wherein the 2′-hydroxyl of the5′-penultimate nucleotide is also methylated. In still otherembodiments, the invention comprises any and all of themodified-nucleotide-capped RNA compositions above having either a cap 0or a cap I structure, wherein the modified-nucleotide-capped RNAadditionally has a poly(A) tail on the 3′ terminus.

Methods and Kits for Using Modified-Nucleotide-Capped RNA for In Vitroand In Vivo Translation

In some embodiments, the present invention provides methods thatcomprise the step of translating the modified-nucleotide-capped RNAhaving either a cap 0 or a cap I structure into protein. Although anunderstanding of the mechanism is not necessary to practice the presentinvention and the present invention is not limited to any particularmechanism of action, in some embodiments, compositions and methods ofthe present invention produce modified-nucleotide-capped RNA (e.g.,mRNA) that is translated more efficiently in vitro or in vivo due to theefficiency at which the capping enzyme system is able to cap uncappedRNA comprising primary RNA transcripts or RNA having a 5′-diphosphate,and/or because of the characteristics of the modified-nucleotide-cappedRNA to serve as a substrate for translation.

In preferred embodiments, the modified-nucleotide-capped RNA that isused for translation has either a cap 0 or a cap I structure and ispolyadenylated. However, since the presence and length of a 3′-poly(A)tail has a greater effect on in vivo translation ofmodified-nucleotide-capped RNA in a eukaryotic cell than on in vitrotranslation of the modified-nucleotide-capped RNA in cell-free extracts,the poly(A) tail length varies in different embodiments. In someembodiments, the poly(A) tail has a length of about 30 nucleotides. Inother embodiments, the poly(A) tail has a length of about 30 nucleotidesto at about 100 nucleotides. In other embodiments, the poly(A) tail hasa length of about 100 nucleotides to at about 200 nucleotides. In stillother embodiments, the poly(A) tail has a length of about 200nucleotides to about 400 nucleotides. In some embodiments, the poly(A)tail has a length of greater than 400 nucleotides. In some embodimentswherein the modified-nucleotide-capped RNA is translated in vitro incell-free extracts, the modified-nucleotide-capped RNA lacks a poly(A)tail.

In some embodiments, the kits and methods use a modified cap nucleotideto synthesize a modified-nucleotide-capped RNA with improved in vitro orin vivo translation properties compared to capped RNA comprising thesame sequence but which is uncapped or which comprises the N⁷-methyl-Gcap nucleotide. For example, but without limitation, in someembodiments, the method or kit uses O⁶-Me-GTP to synthesizemodified-nucleotide-capped RNA that results in improved translation,especially in vivo. In other embodiments, a method or kit of theinvention uses another modified cap nucleotide that results in levels ofin vitro or in vivo translation which are either similar to or higherthan what is obtained with capped RNA comprising the standardN⁷-methyl-G cap nucleotide, in some cases, depending on the cell andother factors. In some embodiments, the modified cap nucleotide providesother benefits in addition to being translated efficiently, such as amethod to capture or label the modified-nucleotide-capped RNA (e.g.,using a modified cap nucleotide that comprises an amino, azido, or thiolgroup).

Thus, in some embodiments, compositions and methods of the presentinvention are used for synthesis of a modified-nucleotide-capped RNA foruse in in vitro translation reactions, or for in vivo translationfollowing transformation or transfection of eukaryotic cells with themodified-nucleotide-capped RNA.

In some embodiments, compositions and methods of the present inventionproduce modified-nucleotide-capped RNA for use in in vitro splicingreactions (e.g., see, Konarska et al., Cell 38: 731-736, 1984; Edery etal., Proc. Natl. Acad. Sci. USA 82: 7590-7594, 1985). In someembodiments, the modified-nucleotide-capped RNAs of the presentinvention are used for functional studies of heterogeneous nuclear RNAsand viral RNAs.

Cell-Free In Vitro Translation of Modified-Nucleotide-Capped RNA

In some embodiments, the invention provides methods for improvedcell-free production of proteins or polypeptides for a variety ofapplications. Without limitation, in some embodiments, cell-freetranslation of modified-nucleotide-capped RNA is used to produceproteins or polypeptides for industrial use (e.g., as enzymes for usefor food processing or in cleaning products, such as laundry cleaningproducts), for use for pharmaceutical bioprocessing, for use formolecular diagnostic enzymes, or for therapeutic use (e.g., for use invaccines that are administered directly to a patient by intradermalinjection or another suitable route, or for use in loading anantigen-presenting cell (APC), such as a dendritic cell, with thepolypeptide).

In some embodiments of the invention, a kit for translatingmodified-nucleotide-capped RNA, besides comprising a capping enzymesystem and a modified cap nucleotide, additionally comprises an in vitrotranslation system. Also, one embodiment of the method of the inventionfurther comprises the step of incubating the modified-nucleotide-cappedRNA having either a cap 0 or a cap I structure in an in vitrotranslation system under conditions wherein protein encoded by themodified-nucleotide-capped RNA is obtained. In some embodiments of thekit or method, the in vitro translation system comprises a cell-freeextract selected from the group consisting of a plant, an animal, and ayeast or fungal cell-free extract. In some embodiments of the kit ormethod, the cell-free extract is selected from the group consisting of awheat germ lysate, a rabbit reticulocyte lysate, a drosophila embryolysate, and a human reticulocyte lysate, wherein the human reticulocytesare derived from human stem cells in culture. In some embodiments, thehuman reticulocytes are prepared from embryonic stem cells. In otherembodiments, the human reticulocytes are prepared from adult stem cellsfrom a patient with a condition. In other embodiments, the humanreticulocytes are prepared from adult stem cells from a donor.

The present invention further provides a method for coupled formation ofmodified-nucleotide-capped RNA and translation of themodified-nucleotide-capped RNA in a cell-free extract. For example, insome embodiments, this coupled formation comprises: (a) contacting anuncapped RNA comprising a primary RNA transcript or RNA having a5′-diphosphate with a modified cap nucleotide and a capping enzymesystem, such as a poxvirus capping enzyme, under conditions permissiveto the formation of modified-nucleotide-capped RNA, thereby forming amodified-nucleotide-capped RNA transcript; and (b) incubating themodified-nucleotide-capped RNA transcript formed in step (a) with acell-free extract under conditions permissive for protein translation.

In some embodiments, the present invention provides a method forcoupling catalyzed formation of modified-nucleotide-capped RNA andtranslation of the modified-nucleotide-capped RNA in a cell-free extractcomprising: (a) contacting an uncapped RNA comprising a primary RNAtranscript or RNA having a 5′-diphosphate with a capping enzyme systemand modified cap nucleotide under conditions wherein amodified-nucleotide-capped RNA is formed; and (b) incubating themodified-nucleotide-capped RNA formed in step (a) with a cell-freeextract under conditions such that protein translation of themodified-nucleotide-capped RNA occurs.

In some embodiments, the present invention provides a method forsequentially coupling RNA transcription and catalyzed formation of amodified-nucleotide-capped RNA and translation of themodified-nucleotide-capped RNA in a cell-free extract comprising: (a)contacting a DNA template with RNA polymerase in a reaction buffer underconditions such that an uncapped RNA transcript is formed; (b)contacting the RNA transcript with a capping enzyme system and modifiedcap nucleotide under conditions wherein a modified-nucleotide-capped RNAis formed; and (c) incubating the modified-nucleotide-capped RNA formedin step (b) with a cell-free extract under conditions wherein proteintranslation of the modified-nucleotide-capped RNA occurs.

Methods and Kits for Using Polypeptides Obtained by In Vitro Translationof Modified-Nucleotide-Capped RNA for Making Vaccines orImmunotherapeutic Products

In some embodiments, the present invention provides methods thatcomprise using the polypeptide obtained from cell-free translation ofmodified-nucleotide-capped RNA to make vaccine for a human or animalpatient in order to prevent or treat a condition. In some embodiments inwhich the modified-nucleotide-capped RNA obtained from capping ofuncapped RNA comprising a primary RNA transcript or RNA having a5′-diphosphate using a capping enzyme system and a modified capnucleotide is translated in vitro, the method further comprises the stepof using the polypeptides obtained as antigens for loading of anantigen-presenting cell (APC), such as a dendritic cell, a macrophage,an epithelial cell, or an artificially generated APC from a human or ananimal, thereby producing a “polypeptide-loaded” (or an“antigen-loaded”) APC that presents on its surface antigenic epitopesencoded by the modified-nucleotide-capped RNA, wherein the epitope iscapable of inducing T cell proliferation. Thus, in some embodiments, themethod additionally comprises the step of contacting the polypeptideobtained from cell-free translation of modified-nucleotide-capped RNAwith an APC, selected from the group consisting of a dendritic cell, amacrophage, an epithelial cell, and an artificially generated APC,whereby a polypeptide-loaded APC is obtained. Thus, the invention alsocomprises a method for producing a polypeptide-loaded antigen presentingcell (APC), said method further comprising introducing the polypeptidesobtained from in vitro translation of polyadenylatedmodified-nucleotide-capped RNA into or in contact with an APC, therebyproducing a polypeptide-loaded APC. In some embodiments, the methodfurther comprises using the polypeptide-loaded APC to make a vaccine toprevent a condition or as an immunotherapy to treat a condition in ahuman or animal patient.

In some embodiments the modified-nucleotide-capped RNA used for in vitrotranslation and for loading the APC is derived from a biologicalspecimen from the patient with the condition (e.g., from the tumor ofthe patient who has the tumor). In some embodiments, themodified-nucleotide-capped RNA used for in vitro translation is derivedfrom a biological specimen from another person with the condition, suchas from a tumor of a donor. In some embodiments, themodified-nucleotide-capped RNA used for in vitro translation is preparedfrom uncapped RNA comprising primary RNA transcripts or RNA having a5′-diphosphate that is obtained following fractionation by subtractivehybridization and RNA amplification. In some embodiments of the method,the uncapped RNA comprising a primary RNA transcript or RNA having a5′-diphosphate that is used to obtain the modified-nucleotide-capped RNAfor in vitro translation is from an in vitro transcription reaction oran RNA amplification reaction that produces sense RNA (e.g., but withoutlimitation, by amplification of RNA from one or a small number of cancercells from a patient). In preferred embodiments of the invention, themodified-nucleotide-capped RNA is prepared from uncapped RNA comprisingprimary RNA transcripts or RNA having a 5′-diphosphate that is obtainedfollowing fractionation of RNA from a biological source by subtractivehybridization and digestion, whereby the uncapped RNA iscondition-specific. In a preferred embodiment, the subtractivehybridization and digestion method used is the cap-dependent subtractionmethod of the present invention. In some embodiments, thecondition-specific RNA is further amplified using a sense RNAamplification reaction.

The invention also comprises kits for making such polypeptide-loaded (orantigen-loaded) APC, including the cells used therefor. By way ofexample, but without limitation, the kit can comprise media and reagentsfor incubating the APC with the polypeptides and growing the cells. Insome embodiments, the kit additionally comprises one or more adjuvantsor other compositions for enhancing or modulating the immune response.

The invention also includes compositions comprising thepolypeptide-loaded (or antigen-loaded) APCs, as well as kits thatadditionally comprise such polypeptide-loaded (or antigen-loaded) APCs,and which may also contain one or more adjuvants or other compositionsfor enhancing or modulating the immune response. Thus, the presentinvention comprises an APC that is loaded with an antigenic polypeptideobtained by in vitro translation of a modified-nucleotide-capped RNAhaving a cap 0 or a cap I structure, either with or without a poly(A)tail, and which APC is capable of producing antigenic epitopes encodedby said modified-nucleotide-capped RNA on its surface. The APC isselected from the group consisting of a dendritic cell, a macrophage, anepithelial cell, or an artificially generated APC from a human or ananimal. In some embodiments, the APC is from the patient with thecondition. In some embodiments, the APC is from a donor who is not thepatient. The invention further provides a method for treating orpreventing a condition, such as a cancer condition or a pathogen-inducedcondition, said method comprising administering to the patient atherapeutically effective amount of the polypeptide-loaded APC, whichpolypeptide is obtained by in vitro translation of saidmodified-nucleotide-capped RNA derived from one or more primary RNAtranscripts of cells having said condition.

The invention also provides a method for producing a cytotoxic Tlymphocyte (CTL), said method comprising: providing a T lymphocyte;contacting said T lymphocyte in vitro with the polypeptide-loaded APCcomprising the polypeptide obtained by in vitro translation of themodified-nucleotide-capped RNA; and maintaining said T lymphocyte underconditions conducive to CTL proliferation, thereby producing a CTL.Thus, one embodiment of the invention is a composition comprising a CTLobtained by contacting the T lymphocyte with the polypeptide-loaded APCcomprising the polypeptide obtained by in vitro translation of themodified-nucleotide-capped RNA, and maintaining said T lymphocyte underconditions conducive to CTL proliferation. In some embodiments, the Tlymphocyte is derived from the patient with the condition. In someembodiments, the T lymphocyte is derived from a donor. The inventionalso includes the CTL produced according to this method. The inventionfurther provides a method for treating or preventing a condition, suchas tumor formation or a pathogen infection, in a patient, said methodcomprising administering to the patient a therapeutically effectiveamount of the CTL obtained using this method.

In some embodiments in which the modified-nucleotide-capped RNA istranslated in vitro, the method further comprises using the polypeptidesobtained as antigens to make a vaccine for administration directly to ahuman or animal patient. In some embodiments, the invention comprises acomposition comprising the polypeptides obtained by in vitro translationof the modified-nucleotide-capped RNA. The composition comprising thepolypeptides can also comprise other compositions, such as adjuvants orother compositions, in order to enhance or modulate the antigenic and/ortherapeutic effect. The composition comprising the polypeptides can beformulated for administration by any route commonly used in the art,such as but not limited to, a formulation for intradermal injection,subcutaneous injection, intravenous injection, transdermal application(e.g., using a “patch”), for use as a nasal spray or for use by anotherroute that is determined to be effective for the particular composition.In some embodiments, the modified-nucleotide-capped RNA for in vitrotranslation is prepared from uncapped RNA derived from primary RNAtranscripts from a patient with a condition, such as a cancer or aninfection with a bacterial, viral or fungal pathogen. Thus, thesecompositions can be used for administering the in vitro-synthesizedpolypeptides to a human or animal patient as a vaccine or immunotherapyin order to prevent or treat a condition.

In Vivo Translation of Modified-Nucleotide-Capped RNA

In still other embodiments the present invention provides methodswherein the modified-nucleotide-capped RNA obtained from capping ofuncapped RNA comprising primary RNA transcripts or RNA having a5′-diphosphate is translated into protein using a capping enzyme systemand a modified cap nucleotide. In some embodiments, the method comprisesthe step of transforming a living eukaryotic cell with themodified-nucleotide-capped RNA, under conditions whereby translation orexpression of protein encoded by the modified-nucleotide-capped RNAoccurs in the eukaryotic cell in vivo.

In some embodiments the uncapped RNA comprising primary RNA transcriptsor RNA having a 5′-phosphate that is used to synthesize themodified-nucleotide-capped RNA for transforming the eukaryotic cells isderived from a biological specimen. In some preferred embodiments, theuncapped RNA that is used to synthesize the modified-nucleotide-cappedRNA for transforming the eukaryotic cells comprises prokaryotic (i.e.,bacterial) mRNA. In some embodiments, the uncapped RNA that is used tosynthesize the modified-nucleotide-capped RNA for transforming theeukaryotic cells is from a biological specimen from a human or animalpatient with a condition (e.g., from the tumor of the patient who hasthe tumor or from a nematode, or bacterial, viral or fungal pathogen, orfrom a eukaryotic cell, tissue, organ or organism infected with thepathogen). In some embodiments, the uncapped RNA that is used tosynthesize the modified-nucleotide-capped RNA for transforming theeukaryotic cells is from a biological specimen from another human oranimal patient with the condition, such as from a tumor of a donor. Insome embodiments, the modified-nucleotide-capped RNA used fortransforming the eukaryotic cells is synthesized from uncapped RNAcomprising primary RNA transcripts or RNA having a 5′-diphosphate thatis obtained following fractionation by subtractive hybridization and RNAamplification. In some embodiments of the method, the uncapped RNAcomprising a primary RNA transcript or RNA having a 5′-diphosphate thatis used to synthesize the modified-nucleotide-capped RNA fortransforming the eukaryotic cells is from an in vitro transcriptionreaction or an RNA amplification reaction. For example, but withoutlimitation, in some embodiments the uncapped RNA is synthesized byamplification of mRNA from one or a small number of cancer cells from apatient. In preferred embodiments of the invention, themodified-nucleotide-capped RNA for transforming the eukaryotic cells issynthesized from uncapped RNA comprising primary RNA transcriptsobtained following fractionation of RNA from a biological source bysubtractive hybridization and digestion, whereby the uncapped RNA iscondition-specific. In a preferred embodiment, the subtractivehybridization and digestion method used is the cap-dependent subtractionmethod of the present invention, described herein. In some embodiments,the condition-specific RNA is further amplified using a sense RNAamplification reaction.

Without limiting the invention, the eukaryotic cell is selected from thegroup consisting of a dendritic cell, a macrophage, an epithelial cell,an artificially generated APC from a human or an animal, an oocyte(e.g., a Xenopus oocyte), a somatic cell of any type from a human, ananimal (e.g., a Chinese hamster ovary cell), a plant, and a fungus,provided that a transformation, transfection, or cell fusion system isknown or can be developed for introducing the modified-nucleotide-cappedRNA into the cell. In some preferred embodiments, the eukaryotic cell isa dendritic cell from a human or an animal. In some embodiments, thestep of transforming the eukaryotic cells comprises microinjection(e.g., into oocytes), transfection, lipofection, electroporation, invivo transposition with a transposome complex, or transformation of theeukaryotic cells by another means known in the art.

In some embodiments of this aspect of the invention, themodified-nucleotide-capped RNA has a cap 0 structure. In some preferredembodiments, the modified-nucleotide-capped RNA has a cap I structure.In some preferred embodiments, the modified-nucleotide-capped RNA havingeither a cap 0 or a cap I structure has a poly(A) tail on the3′-terminus. In some preferred embodiments, themodified-nucleotide-capped RNA used to transform or transfect theeukaryotic cell has a poly(A) tail on the 3′-terminus and a 2′-O-methylgroup on the 5′-penultimate nucleotide (i.e., the cap of themodified-nucleotide-capped RNA has a cap I structure). In someembodiments, the modified-nucleotide-capped RNA that is used totransform or transfect the eukaryotic cell comprises uncapped RNA thathas been capped with a capping enzyme system using a modified capnucleotide, including but not limited to a modified cap nucleotideselected from the group consisting of: O⁶-Me-GTP; 2′-amino-2′-dGTP;2′-azido-2′-dGTP; 2′-fluoro-2′-dGTP; 2′-OMe-GTP; 3′-amino-3′-dGTP;3′-azido-3′-dGTP; 3′-fluoro-3′-dGTP; 3′-OMe-GTP; and 3′-dGTP. In someembodiments wherein the modified-nucleotide-capped RNA comprises apoly(A) tail or a cap I structure, the modified-nucleotide-capped RNA issynthesized using a capping enzyme system and 2′-dGTP as the modifiedcap nucleotide. In one embodiment, the modified-nucleotide-capped RNAhas an N⁷-methyl-O⁶-methylguanosine modified cap nucleoside, which wasfound in some experiments to provide higher in vivo translationefficiency than the same RNA with an unmodified m⁷G cap nucleotide. Inother embodiments, the modified-nucleotide-capped RNA has a differentmodified cap nucleotide.

Methods and Kits for Using Modified-Nucleotide-Capped RNA for MakingVaccines or Immunotherapeutic Products

In some embodiments the present invention provides methods wherein themodified-nucleotide-capped RNA is translated in vivo, the eukaryoticcell is a human or animal cell of a patient and said cell is transformedby the modified-nucleotide-capped RNA having a poly(A) tail by directinjection into the patient, such as by intradermal injection of a humanpatient as described by Carralot, J-P, et al. (Genetic Vaccines andTherapy 3: 6, 2005). Thus, in some embodiments, themodified-nucleotide-capped RNA comprises a composition for use as avaccine for treating or preventing a condition, such as a cancercondition or a pathogen-induced condition. In some embodiments, theinvention further comprises using the modified-nucleotide-capped RNAhaving a poly(A) tail to make a composition comprising a vaccine, whichvaccine can be administered to patients as one or more injections of atherapeutically effective amount. In some embodiments, the compositioncomprises polyadenylated modified-nucleotide-capped RNA derived fromprimary RNA transcripts amplified using cDNA prepared fromtumor-specific or pathogen-specific mRNA.

In some other embodiments of the method wherein themodified-nucleotide-capped RNA is translated in vivo in a eukaryoticcell, the modified-nucleotide-capped RNA has either a cap 0 or a cap Istructure and is polyadenylated and the eukaryotic cell is anantigen-presenting cell (APC) selected from the group consisting of adendritic cell, a macrophage, an epithelial cell, or an artificiallygenerated APC from a human or an animal. In some embodiments, the methodfurther comprises the step of using the modified-nucleotide-capped RNAto make an RNA-loaded APC for use as a vaccine. In a preferredembodiment, the RNA-loaded APC is a dendritic cell. With respect to themethods and kits of the present invention for preparing an RNA-loadedAPC using a modified-nucleotide-capped RNA, and for using saidRNA-loaded APC as a vaccine, methods and compositions known in the artwith respect to other RNA molecules can be used, as described in U.S.Patent Application Nos. 20020018769; and 20060057130; and in U.S. Pat.Nos. 6,670,186; 6,387,701; 6,306,388; 5,853,719; and 5,831,068, and inBoczkowski, D et al., J. Exp. Med. 184: 465-472, 1996; Heiser, A et al.,J. Clinical Investigation 109: 409-417, 2002; Su, Z et al., Cancer Res.63: 2127-2133, 2003; Gilboa, E and Vieweg, J, Immunol. Rev. 199:251-263, 2004; Harris, J et al., BBA 1724: 127-136, 2005; and Mockey, Met al. (Biochem. Biophys. Res. Comm. 340: 1062, 2006).

In some embodiments, the modified-nucleotide-capped RNA encodes a tumorantigen. In some embodiments, the modified-nucleotide-capped RNA encodesan antigen against a pathogen, such as a bacterial, viral or fungalantigen, or an antigen induced in a human or animal upon infection bysuch pathogen. Thus, some embodiments of the invention comprise a methodfor producing an RNA-loaded APC that presents on its surface a tumorantigenic epitope or a pathogen antigenic epitope encoded by the RNA,wherein the epitope induces T cell proliferation, said method furthercomprising the step of introducing the polyadenylatedmodified-nucleotide-capped RNA encoding the antigens into anantigen-presenting cell. By “RNA-loaded” antigen-presenting cell ismeant an APC (e.g., a macrophage or dendritic cell) that was incubatedor transfected with RNA (e.g., RNA derived from a tumor or pathogen orfrom eukaryotic cells infected by a pathogen). Such RNA can be loadedinto the APC by using conventional nucleic acid transfection methods,such as lipid-mediated transfection, electroporation, and calciumphosphate transfection. For example, but without limitation,modified-nucleotide-capped RNA can be introduced into an APC byincubating the APC with the modified-nucleotide-capped RNA (or extract)for 1 to 24 hours (e.g., 2 hours) at 37° C., preferably in the presenceof a cationic lipid.

In some embodiments of the method, the uncapped RNA comprising primaryRNA transcripts or RNA having a 5′-diphosphate used to obtain thepolyadenylated modified-nucleotide-capped RNA is transcribed from clonesin a cDNA library prepared from amplified tumor mRNA (or frompathogen-specific mRNA). In some embodiments, themodified-nucleotide-capped RNA is transcribed from clones in a cDNAlibrary prepared as described by Carralot J-P, et al. (Genetic Vaccinesand Therapy 3: 6, 2005). In preferred embodiments of the invention, thepolyadenylated modified-nucleotide-capped RNA is prepared from uncappedRNA comprising primary RNA transcripts or RNA having a 5′-diphosphateobtained following fractionation by subtractive hybridization, digestionand RNA amplification of RNA from a biological source, whereby in vivotranslation of the polyadenylated modified-nucleotide-capped RNA in theAPC results in presentation of only condition-specific antigens by theAPC. In some embodiments, the polyadenylated modified-nucleotide-cappedRNA is prepared from RNA obtained by subtractive hybridization anddigestion. In some embodiments, the subtractive hybridization,digestion, and RNA amplification is performed as described in U.S. Pat.No. 5,712,127. In other preferred embodiments, the polyadenylatedmodified-nucleotide-capped RNA is prepared from RNA obtained bysubtractive hybridization and digestion using the cap-dependentsubtraction method of the present invention. In some embodiments, theuncapped RNA that is used to synthesize the modified-nucleotide-cappedRNA is amplified using an in vitro transcription reaction or RNAamplification reaction. In some embodiments, the uncapped RNA isamplified using the RNA amplification method described in U.S. PatentApplication No. 20050153333 of Sooknanan. In some embodiments, thesubtracted and amplified uncapped RNA comprises RNA from a condition(e.g., a tumor cell condition) from which RNA that is also present in anormal cell of the same type has been removed by subtractivehybridization and the remaining RNA is then amplified using an RNAamplification reaction. Thus, in these embodiments, the polyadenylatedmodified-nucleotide-capped RNA made using the uncapped RNA comprisingprimary RNA transcripts or RNA having a 5′-diphosphate from this RNAamplification reaction is condition-specific (e.g., tumor-specific), andthe APC (e.g., dendritic cell) also translates or expressescondition-specific (e.g., tumor-specific) polypeptides encoded by thisRNA. In some embodiments of the invention, the polyadenylatedmodified-nucleotide-capped RNA is prepared using RNA derived from cancercells. In other embodiments of the invention, the polyadenylatedmodified-nucleotide-capped RNA is prepared using RNA derived from apathogen, such as a bacterial, viral or fungal pathogen, or from aeukaryotic cell that is infected by a bacterial, viral or fungalpathogen, whereby the APC translates or expresses polypeptides that arepathogen-specific. Thus, the invention also comprises a method forproducing an RNA-loaded antigen presenting cell (APC), said methodfurther comprising introducing into the APC the condition-specificpolyadenylated modified-nucleotide-capped RNA, thereby producing anRNA-loaded APC. In some embodiments of this method, thecondition-specific polyadenylated modified-nucleotide-capped RNA isselected from the group consisting of tumor-specific RNA andpathogen-specific RNA. The invention also comprises kits for performingsuch methods wherein the modified-nucleotide-capped RNA is translated invivo following transformation of a living eukaryotic cell. For example,in some embodiments, the kit additionally comprises the eukaryotic celland, optionally, transformation or transfection reagents (e.g., thecationic lipid DOTAP or 1:1 (w/w) DOTMA:DOPE (i.e., LIPOFECTIN) or othertransfection reagents known in the art that are appropriate for theparticular cell type). In some embodiments, the eukaryotic cell in thekit is an oocyte (e.g., a Xenopus oocyte), or a somatic cell of any typefrom a human, an animal, a plant, or a fungus. In some embodiments, theeukaryotic cell in the kit is an APC selected from the group consistingof a dendritic cell, a macrophage, an epithelial cell, and anartificially generated APC from a human or an animal. The presentinvention also comprises a eukaryotic cell that is transformed ortransfected with a modified-nucleotide-capped RNA having a cap 0 or acap I structure, including a polyadenylated modified-nucleotide-cappedRNA. In some embodiments, the invention comprises an APC, including adendritic cell, a macrophage, an epithelial cell, or an artificiallygenerated APC from a human or an animal, that is transformed ortransfected with a modified-nucleotide-capped RNA having a cap 0 or acap I structure, including a polyadenylated modified-nucleotide-cappedRNA, and which APC is capable of producing antigenic epitopes encoded bysaid modified-nucleotide-capped RNA on its surface. The inventionfurther provides a method for treating or preventing a condition in ahuman or animal, such as a cancer condition or a pathogen-inducedcondition, said method comprising administering to the patient atherapeutically effective amount of the RNA-loaded APC obtained byintroduction into said APC of the polyadenylatedmodified-nucleotide-capped RNA prepared from uncapped RNA derived fromprimary RNA transcripts from cells having said condition. In someembodiments, the APC is from the patient with the condition. In someembodiments, the APC is from a donor who is not the patient. In someembodiments the modified-nucleotide-capped RNA is derived from abiological specimen from the patient with the condition (e.g., from thetumor of the patient who has the tumor). In some embodiments, themodified-nucleotide-capped RNA is from a biological specimen fromanother person with the condition. The invention also provides a methodfor producing a cytotoxic T lymphocyte (CTL), said method comprising:providing a T lymphocyte; contacting said T lymphocyte in vitro with theRNA-loaded APC comprising the modified-nucleotide-capped RNA; andmaintaining said T lymphocyte under conditions conducive to CTLproliferation, thereby producing a CTL. The invention also includes theCTL produced according to this method. In some embodiments, the Tlymphocyte is derived from a donor. In some embodiments, the uncappedRNA comprising primary RNA transcripts or RNA having a 5′-diphosphatethat is used to make the condition-specific (e.g., tumor-specific orpathogen-specific) modified-nucleotide-capped RNA for loading the APC isderived from the patient with the condition that receives thistreatment. In some embodiments, the uncapped RNA comprising primary RNAtranscripts or RNA having a 5′-diphosphate that is used to make thecondition-specific modified-nucleotide-capped RNA for loading the APC isderived from a donor. The invention further provides a method fortreating or preventing a condition, such as tumor formation or apathogen infection, in a human or animal patient, said method comprisingadministering to the patient a therapeutically effective amount of theCTL obtained using this method. In some embodiments, the T lymphocyte isderived from the patient with the condition.

Some embodiments of the invention comprise a eukaryotic cell thatcontains a modified-nucleotide capped RNA synthesized using a method ofthe present invention, or a eukaryotic cell that contains a peptide thatis translated from the modified-nucleotide capped RNA. In someembodiments, the eukaryotic cell that contains themodified-nucleotide-capped RNA having either a cap 0 or a cap Istructure, or with our without a poly(A) tail, is an oocyte (e.g., aXenopus oocyte), or a somatic cell of any type from a human, an animal,a plant, or a fungus. In some embodiments, the eukaryotic cell is anAPC, such as, but not limited to a dendritic cell, a macrophage, anepithelial cell, or an artificially generated APC from a human or ananimal. In some embodiments, the modified-nucleotide-capped RNA encodesa tumor antigen (i.e., a tumor-specific antigen) or an antigen from abacterial, viral or fungal pathogen or from a eukaryotic cell that isinfected by a bacterial, viral or fungal pathogen (i.e., apathogen-specific antigen). Thus, some embodiments of the inventioncomprise a modified-nucleotide-capped RNA-loaded antigen-presenting cell(APC) that presents on its surface a tumor-specific antigenic epitope ora pathogen-specific antigenic epitope encoded by the RNA, wherein theepitope induces T cell proliferation.

Methods and Kits for In Vivo Expression of Prokaryotic Bacterial mRNA inEukaryotes

The present invention also provides methods for synthesizing amodified-nucleotide-capped RNA by capping of uncapped RNA comprisingprimary RNA transcripts or RNA having a 5′-diphosphate using a cappingenzyme system and a modified cap nucleotide that provides a method forexpressing one or more prokaryotic (e.g., bacterial) mRNA transcripts ina eukaryotic cell. Thus, in some preferred embodiments, the method isused to express or screen for expression of prokaryotic mRNA thatencodes a desired enzymatic activity or polypeptide in a eukaryoticcell, said method comprising: (i) providing a sample containingprokaryotic mRNA, a capping enzyme system, a modified cap nucleotide,and a eukaryotic cell; (ii) contacting the prokaryotic mRNA with thecapping enzyme system and the modified cap nucleotide under conditionswherein modified-nucleotide-capped RNA is synthesized; (iii)transforming the eukaryotic cell with the modified-nucleotide-capped RNAand incubating said transformed eukaryotic cell in a medium and underconditions that sustain said eukaryotic cell; and (iv) detecting orscreening for the presence of the enzymatic activity or polypeptide inthe eukaryotic cell. However, the invention is not limited to the use ofa modified cap nucleotide for expressing prokaryotic mRNA in aeukaryotic cell. The method can be used with any cap nucleotide that isa substrate for the capping enzyme system, including a modified capnucleotide or a cap nucleotide that is not a modified cap nucleotide(e.g., GTP). Thus, in some preferred embodiments, the method is used toexpress or screen for expression of prokaryotic mRNA that encodes adesired enzymatic activity or polypeptide in a eukaryotic cell, saidmethod comprising: (i) providing a sample containing prokaryotic mRNA, acapping enzyme system, a cap nucleotide, and a eukaryotic cell; (ii)contacting the prokaryotic mRNA with the capping enzyme system and thecap nucleotide under conditions wherein capped RNA is synthesized; (iii)transforming a eukaryotic cell with the modified-nucleotide-capped RNAand incubating said transformed eukaryotic cell in a medium and underconditions that sustain said eukaryotic cell; and (iv) detecting orscreening for the presence of the enzymatic activity or polypeptide inthe eukaryotic cell. Thus, the present invention encompasses the use ofa cap nucleotide that is not a modified cap nucleotide in all of theembodiments of the present invention that comprise expressing aprokaryotic primary RNA (e.g., bacterial or mycoplasmal mRN) in aeukaryotic cell, unless said embodiment requires the use of a modifiedcap nucleotide that comprises a chemical moiety for capture or labelingof the modified cap nucleotide. In some embodiments of the above methodsfor expressing a prokaryotic primary RNA in a eukaryotic cell, themodified-nucleotide-capped RNA (or capped RNA) synthesized using themethod has a cap 0 structure, whereas in other embodiments, themodified-nucleotide-capped RNA (or capped RNA) synthesized has a cap Istructure (i.e., it has a 2′-O-methyl group on the penultimatenucleotide. In preferred embodiments, the modified-nucleotide-capped RNA(or capped RNA) that is used to transform the eukaryotic cell in step(ii) additionally comprises a poly(A) tail on the 3′-terminus. Themethod provides a powerful way to identify and screen for prokaryoticmRNA having a desired enzymatic activity or effect in a eukaryotic cell,or for producing a polypeptide in the eukaryotic cell that has abeneficial effect or useful application (e.g., for expressing an antigenin an antigen-presenting cell loaded with the modified-nucleotide-cappedRNA (or capped RNA), as described elsewhere herein. For example, butwithout limitation, the method can be used for screening all prokaryoticmRNA from one or more prokaryotic organisms (e.g., bacterium) for thedesired enzymatic activity or effect.

In this embodiment, the uncapped RNA that is capped using the cappingenzyme system comprises one or more prokaryotic mRNA transcripts (e.g.,bacterial mRNA), and the method additionally comprises transforming aeukaryotic cell with the modified-nucleotide-capped RNA and assaying orscreening for the activity or effects of the proteins encoded by saidone or more prokaryotic mRNA transcripts in the eukaryotic cell. In someembodiments, the modified-nucleotide-capped RNA that is used totransform the eukaryotic cell comprises prokaryotic bacterial mRNA thatis capped with a capping enzyme system using a modified cap nucleotidecomprising a modified 2′- or 3′-deoxyguanosine-5′-triphosphate whereinthe respective 2′- or 3′-deoxy position of the sugar moiety issubstituted by a moiety comprising an amino group, an azido group, afluorine group, or a methoxy group, or a modifiedguanosine-5′-triphosphate wherein the O6 oxygen of the guanine base ismodified by being substituted with an alkyl group. In some embodiments,the modified cap nucleotide is selected from the group consisting ofO⁶-Me-GTP, 2′-amino-2′-dGTP, 2′-azido-2′-dGTP, 2′-fluoro-2′-dGTP,2′-OMe-GTP, 3′-amino-3′-dGTP, 3′-OMe-GTP, and 3′-dGTP. In someembodiments wherein the modified-nucleotide-capped RNA comprises apoly(A) tail or has a cap I structure, the modified cap nucleotide is2′-dGTP. The invention is also not limited to use of a modified capnucleotide or to use of a modified-nucleotide-capped RNA. Thus, in someembodiments, any nucleotide (e.g., GTP) that is a substrate for thecapping enzyme system is used to synthesize capped prokaryotic mRNAhaving a poly(A) tail for screening for expression of prokaryotic mRNAin a eukaryotic cell. In preferred embodiments that comprisesynthesizing modified-nucleotide-capped RNA, themodified-nucleotide-capped RNA comprising prokaryotic bacterial mRNAthat is used to transform the eukaryotic cell has a 3′-poly(A) tail. Insome embodiments, which are preferred, the 3′-polyadenylatedmodified-nucleotide-capped RNA comprising prokaryotic bacterial mRNAthat is used to transform the eukaryotic cell additionally has a2′-O-methyl group on the 5′-penultimate nucleotide (i.e., the cap of themodified-nucleotide-capped RNA has a cap I structure). In someembodiments, the prokaryotic mRNA that is used to synthesize themodified-nucleotide-capped RNA is first amplified using an in vitrotranscription or RNA amplification reaction. In some embodiments, theprokaryotic mRNA is fractionated using subtractive hybridization,digestion, and RNA amplification. In some preferred embodiments, theprokaryotic mRNA is fractionated using the cap-dependent subtractionmethod of the present invention. In some such embodiments, a poly(A)tail is synthesized on the 3′-terminus of amplified prokaryotic mRNA orthe modified-nucleotide-capped RNA by in vitro transcription of a DNAtemplate that encodes the prokaryotic mRNA and the poly(A) tail. Theinvention also comprises a composition of a modified-nucleotide-cappedRNA comprising prokaryotic bacterial mRNA that is made using any of themethods of the invention.

Based on the above description, those with knowledge in the art willknow how to use the method for a variety of applications and formats.The method can be used to screen mRNA from any of the vast diversity ofprokaryotic organisms, whether previously cultured or not, forexpression of protein activities that may be beneficial in a eukaryotefor therapeutic or commercial applications. For example, but withoutlimitation, the present invention contemplates using the method toscreen for expression of prokaryotic mRNA in eukaryotes in order toidentify a protein activity that will effectively complement a proteinactivity that is absent or deleted or mutated so as to result in adisease, susceptibility or metabolic problem in a human or othereukaryote. In other embodiments, the method is used to screen for anenzymatic activity that is absent in a metabolic pathway that isexpressed in a eukaryotic cell. In some embodiments, the enzymaticactivity of the metabolic pathway that is absent is an enzymaticactivity encoded by a gene that is mutated in the eukaryotic cell. Inother embodiments, the metabolic pathway comprises genes from anotherorganism that are expressed in the eukaryotic cell and the gene thatencodes the missing enzymatic activity is not present or is notsufficiently expressed in the eukaryotic cell. In some embodiments, themetabolic pathway comprising genes from another organism that isexpressed in the eukaryotic cell is expressed from genes that are clonedin a vector, such as a plasmid, cosmid, BAC, fosmid, transposon, phage,or other DNA vector. Still further, in some embodiments, the presentinvention contemplates using the method to screen for expression ofprokaryotic mRNA in a eukaryote in order to identify a protein activitythat will be useful for a commercial process, such as to provide anenzymatic activity that will more efficiently convert specific types ofbiomass to ethanol, n-butanol or other desired chemical compounds. Onceprimary prokaryotic mRNA transcripts are identified with the desiredactivity, cDNA corresponding to the mRNA that encodes the activity iscloned, genetically engineered, and expressed in the appropriate fungal,plant or other eukaryotic organism for the intended commercial purpose.Although much work has been carried out in the prior art on using DNAfrom prokaryotes in order to identify genes with useful activities,little or no work has been performed to use prokaryotic mRNA transcriptsto identify such useful activities. The present invention providesmethods for doing so, which the present inventors contemplate are muchmore efficient and productive than previous methods of screening for DNAfor useful activities. This is because modified-nucleotide-capped andpolyadenylated prokaryotic bacterial mRNA, prepared using the methods ofthe present invention, is likely to be expressed in eukaryotes withoutadditional modification of the mRNA, whereas DNA requires complicatedadditional genetic manipulations, such as addition of appropriatepromoters, expression of tRNAs optimal for codon usage in the organism,and use of other regulatory elements in order to obtain expression ofthe gene in the eukaryote.

It is not currently known in the art to use a capping enzyme system anda cap nucleotide to make a composition comprising capped prokaryoticmRNA, or to additionally make a composition comprising cappedprokaryotic mRNA having a poly(A) tail on its 3′-terminus for expressingsaid capped and polyadenylated prokaryotic mRNA in a eukaryotic cell.Therefore, the methods and kits of the invention for expressingprokaryotic (e.g., bacterial) mRNA in a eukaryotic cell are not limitedto the use of a modified cap nucleotide or use of amodified-nucleotide-capped RNA. In some embodiments, any nucleotide(e.g., GTP) that is a substrate for the capping enzyme system is used tosynthesize capped prokaryotic RNA that is used in the method totransform the eukaryotic cell and to screen for expression. Thus, theinvention further provides a general method for expressing prokaryotic(e.g., bacterial) mRNA in a eukaryotic cell and screening theprokaryotic mRNA for expression of a desired enzymatic activity orstructural protein in a eukaryotic cell, said method comprising: (i)providing an uncapped prokaryotic RNA comprising primary RNA transcriptsor RNA having a 5′-diphosphate, a capping enzyme system, and a capnucleotide comprising a nucleotide that can be used by the cappingenzyme system to cap the uncapped RNA; (ii) contacting the prokaryoticRNA with the capping enzyme system and the cap nucleotide underconditions wherein capped prokaryotic RNA is synthesized; (iii) treatingthe capped prokaryotic RNA under conditions wherein capped prokaryoticRNA having a poly(A) tail on its 3′-terminus is obtained; (iv)transforming a eukaryotic cell with the capped prokaryotic RNA having apoly(A) tail and incubating said transformed eukaryotic cell in a mediumand under conditions that sustain viability; and (v) screening for thepresence of the enzymatic activity or protein in the eukaryotic cell. Insome embodiments, the step of treating the capped prokaryotic RNA instep (iii) comprises contacting the prokaryotic RNA or the cappedprokaryotic RNA with poly(A) polymerase and ATP under conditions whereinthe poly(A) tail is synthesized on its 3′-terminus. In some preferredembodiments, the prokaryotic RNA is bacterial mRNA. In some embodiments,the prokaryotic RNA that is used to synthesize the capped prokaryoticRNA is first amplified using an in vitro transcription or RNAamplification reaction. In some embodiments, the prokaryotic RNA isfractionated using subtractive hybridization, digestion, and RNAamplification. In some preferred embodiments, the prokaryotic RNA isfractionated using the cap-dependent subtraction method of the presentinvention. In some such embodiments, a poly(A) tail is synthesized onthe 3′-terminus of amplified prokaryotic RNA or amplified and cappedprokaryotic RNA by in vitro transcription of a DNA template that encodesthe amplified prokaryotic RNA and the poly(A) tail. In some embodiments,the invention also comprises a composition of a capped prokaryotic RNAhaving a poly(A) tail that is made using any of the methods of theinvention.

The invention further comprises use of any and all methods of theinvention in human or animal cells or in vivo in animals for researchpurposes in order to investigate the effects of such method forpurposes, such as, but not limited to, therapeutic purposes.

Methods and Kits for Cap-Dependent Capture (CDC) and Cap-DependentSubtraction (CDS): Capture, Isolation, Purification, and Subtraction ofUncapped RNA A. Summary of Cap-Dependent Capture (CDC)

One embodiment of the invention provides a method for capturing,isolating and/or purifying uncapped RNA comprising primary RNA or RNAhaving a 5′-diphosphate in a sample. Thus, one embodiment is a methodfor “cap-dependent capture” (“CDC”) of uncapped RNA comprising primaryRNA transcripts or RNA having a 5′-diphosphate, the method comprising:(i) providing a sample comprising the uncapped RNA; a capping enzymesystem; a modified cap nucleotide, wherein the modified cap nucleotidecontains a chemical binding moiety to facilitate binding to anaffinity-tag-binding molecule; and a surface, to which theaffinity-tag-binding molecule is attached; (ii) contacting the uncappedRNA with the capping enzyme system and the modified cap nucleotide underconditions wherein modified-nucleotide-capped RNA is synthesized; (iii)contacting the modified-nucleotide-capped RNA with reagents and underconditions that facilitate binding of the modified cap nucleotide to thesurface to which the affinity-tag-binding molecule is attached; and (iv)contacting the modified-nucleotide-capped RNA to the surface to whichthe affinity-tag-binding molecule is attached under conditions whereinthe modified-nucleotide-capped RNA is bound to the surface, therebycapturing the modified-nucleotide-capped RNA. In some embodiments of thecap-dependent capture method, the chemical binding moiety of themodified cap nucleotide provided in step (i) comprises an affinity tagthat is capable of binding the affinity-tag-binding molecule and step(iii) comprises incubating the modified-nucleotide-capped RNA in abuffer and under conditions that facilitate binding of the modified capnucleotide to surface to which the affinity-tag-binding molecule isattached. In some other embodiments of the cap-dependent capture method,the modified cap nucleotide provided in step (i) contains a chemicalbinding moiety comprising an amino, an azido, or a thiol group and step(iii) comprises contacting the modified-nucleotide-capped RNA with anaffinity tag reagent under conditions wherein the affinity tag ischemically joined to the chemical binding moiety, and further incubatingthe modified-nucleotide-capped RNA in a buffer and under conditions thatfacilitate binding of the modified cap nucleotide to the surface towhich the affinity-tag-binding molecule is attached. Thus, oneembodiment of the invention comprises a kit for cap-dependent capture(CDC), the kit comprising: (i) a capping enzyme system; (ii) a modifiedcap nucleotide, wherein the modified cap nucleotide contains a chemicalbinding moiety to facilitate binding to an affinity-tag-bindingmolecule; and (iii) a surface, to which the affinity-tag-bindingmolecule is attached. In some embodiments of the kit for CDC, thechemical binding moiety of the modified cap nucleotide comprises anaffinity tag that is capable of binding the affinity-tag-bindingmolecule. In other embodiments of the kit for CDC, the chemical bindingmoiety of the modified cap nucleotide comprises an amino, an azido, or athiol group, and the kit additionally comprises: (iv) an affinity tagreagent that is capable of reacting with the respective amino, azido, orthiol group of the modified cap nucleotide, thereby chemically joiningthe affinity tag to the chemical binding moiety of the modified capnucleotide. In preferred embodiments of the methods and kits forcap-dependent capture, the affinity tag is biotin and theaffinity-tag-binding molecule is streptavidin or avidin.

In some preferred embodiments of the cap-dependent capture method, thesample comprising the uncapped RNA also comprises other nucleic acidswhich are not primary RNA transcripts or RNA having a 5′-diphosphate,and method further comprises the step of: (v) separating themodified-nucleotide-capped RNA that is bound to the surface from theother nucleic acids which are not primary RNA transcripts or RNA havinga 5′-diphosphate. In some preferred embodiments, the step of separatingthe modified-nucleotide-capped RNA that is bound to the surface from theother nucleic acids which are not primary RNA transcripts or RNA havinga 5′-diphosphate comprises washing the surface to which themodified-nucleotide-capped RNA is bound. Thus, in some embodiments ofthe kit for CDC, the kit additionally comprises: (v) a solution forwashing the surface to which the modified-nucleotide-capped RNA isbound.

In some embodiments, the method for cap-dependent capture furthercomprises the step of: (vi) contacting the modified-nucleotide-cappedRNA that is bound to the surface with a protein or biochemical reagentunder conditions wherein the triphosphate between the modified capnucleotide and the 5′-penultimate nucleotide of themodified-nucleotide-capped RNA is cleaved, thereby de-capping themodified-nucleotide-capped RNA and releasing the de-capped RNAtherefrom. In some preferred embodiments, the protein or biochemicalreagent that cleaves the triphosphate is a pyrophosphatase or decappingenzyme. In some preferred embodiments, the protein or biochemicalreagent that cleaves the triphosphate is selected from the groupconsisting of tobacco acid pyrophosphatase, Saccharomyces cerevisiaedecapping enzyme, and human decapping enzyme. Thus, the method providesa substantially purified de-capped RNA from the capturedmodified-nucleotide-capped RNA that is bound to the surface. Thesubstantially purified de-capped RNA is useful for additional analysisand use. Thus, in some embodiments of the kit for CDC, the kitadditionally comprises: (vi) a protein or biochemical reagent that iscapable of specifically cleaving the triphosphate in themodified-nucleotide-capped RNA between the modified cap nucleotide andthe 5′-penultimate nucleotide of the modified-nucleotide-capped RNAwithout cleaving other positions in the modified-nucleotide-capped RNA.In some preferred embodiments of the kit, the protein or biochemicalreagent that is capable of specifically cleaving the triphosphateselected from the group consisting of tobacco acid pyrophosphatase,Saccharomyces cerevisiae decapping enzyme, and human decapping enzyme.

Thus, method for cap-dependent capture (CDC) of uncapped RNA is anembodiment of the basic method comprising contacting an uncapped RNAcomprising primary RNA transcripts or RNA having a 5′-diphosphate with acapping enzyme system and a modified cap nucleotide under conditionwherein the uncapped RNA is capped and a modified-nucleotide-capped RNAis synthesized, the method further comprising capturing themodified-nucleotid-capped RNA synthesized. In these embodiments, themethod relies on binding the modified cap nucleotide, which requiresthat it can be specifically joined through a chemical moiety on the capnucleotide to an affinity tag. Thus, in some embodiments of this method,the modified cap nucleotide comprises a guaninenucleoside-5′-triphosphate wherein the 2′- or 3′-position of the sugarcomprises an amino, an azido, or a thiol group, or the 6-position of theguanine base is a thiol, and the method further comprises the step ofcontacting the modified cap nucleotide or the modified-nucleotide-cappedRNA synthesized with the affinity tag reagent under conditions whereinthe affinity tag is chemically joined to the respective amino, azido, orthiol group thereof. In some embodiments of the cap-dependent capturemethod or kit, the modified cap nucleotide comprises: (a) 2′- and/or3′-deoxyguanosine-5′-triphosophate having a 2′ or 3′ substituentconsisting of an amino, an azido, or a thiol group; or (b) a6-thioguanine nucleoside-5′-triphosphate consisting of 6-thio-GTP,6-thio-2′-dGTP, or 6-thio-3′-dGTP. In some preferred embodiments of themethod or kit, the modified cap nucleotide is selected from among:2′-amino-2′-dGTP; 3′-amino-3′-dGTP; 2′-azido-2′-dGTP; 3′-azido-3′-dGTP;2′-mercapto-2′-dGTP (i.e. 2′-SH-2′-dGTP); 3′-mercapto-3′-dGTP;2′-amino-2′,3′-ddGTP 3′-amino-2′,3′-ddGTP; 2′-azido-2′,3′-ddGTP;3′-azido-2′,3′-ddGTP; 2′-mercapto-2′,3′-dGTP; 3′-mercapto-2′,3′-ddGTP;6-thio-GTP (i.e., 6-mercapto-GTP); 6-thio-2′-dGTP; and 6-thio-3′-dGTP.Thus, in some embodiments of the method, a composition comprisingmodified-nucleotide-capped RNA having the affinity tag is obtained,which compositions are also part of the invention.

The cap-dependent capture methods can be used for capturing, isolatingand/or purifying any uncapped RNA comprising primary RNA or RNA having a5′-diphosphate in a sample, including such uncapped RNA that is in asample that also includes other nucleic acids that are not primary RNAtranscripts or RNA having a 5′-diphosphate.

B. Summary of Cap-Dependent Subtraction (CDS)

In some embodiments wherein the method for cap-dependent capture (CDC)is used, the method additionally comprises steps for subtracting (orremoving) those RNAs that are present in or derived from a first samplecomprising one or more cells of a first type or condition that are alsopresent in a second sample comprising one or more cells of a second typeor condition; thus, this method, which is referred to herein as“cap-dependent subtraction” or “CDS”, is useful for obtaining a“subtraction library” of RNA molecules that are only present in thefirst sample, but are not present in the second sample. The method forcap-dependent subtraction comprises: (i) using the method ofcap-dependent capture (CDC) to capture modified-nucleotide-capped RNAcomprising RNA derived from a first sample comprising one or more cellsof a first type or condition on a surface; (ii) contacting RNA derivedfrom a second sample comprising one or more cells of a second type orcondition with one or more primers that anneal to the RNA and anRNA-dependent DNA polymerase under conditions wherein first-strand cDNAthat is complementary to the RNA from the second sample is synthesized,provided that, the first-strand cDNA has such a polarity orcomplementarity that, if the same RNA is present in the first sample, itwill be complementary to the first-strand cDNA prepared from the RNAderived from the second sample; and (iii) contacting the capturedmodified-nucleotide-capped RNA comprising RNA derived from the firstsample with the first-strand cDNA prepared from the RNA derived from thesecond sample under conditions wherein the first-strand cDNA from thesecond sample anneals to complementary capturedmodified-nucleotide-capped RNA from the first sample; and (iv)contacting the nucleic acids in step (iii) with RNase H under conditionswherein RNA that is annealed to DNA is digested, thereby subtractingfrom the captured modified-nucleotide-capped RNA derived from the firstsample those modified-nucleotide-capped RNA molecules or nucleic acidsequences that are also present in the RNA derived from the secondsample. Thus, one embodiment of the invention comprises a kit forcap-dependent subtraction (CDS), the kit comprising: (i) the componentsof the kit for cap-dependent capture, including the capping enzymesystem, the modified cap nucleotide, and the surface, to which theaffinity-tag-binding molecule is attached; and additionally comprising:(ii) an RNA-dependent DNA polymerase and one or more primers forsynthesis of first-strand cDNA derived from the second sample; and (iii)RNase H. In some embodiments of the components for CDC, the chemicalbinding moiety of the modified cap nucleotide comprises an affinity tagthat is capable of binding the affinity-tag-binding molecule. In otherembodiments of the components for CDC, the chemical binding moiety ofthe modified cap nucleotide comprises an amino, an azido, or a thiolgroup, and the kit additionally comprises the affinity tag reagent thatis capable of reacting with the respective amino, azido, or thiol groupof the modified cap nucleotide, thereby chemically joining the affinitytag to the chemical binding moiety of the modified cap nucleotide. Inpreferred embodiments of the methods and kits for cap-dependentsubtraction, the affinity tag is biotin and the affinity-tag-bindingmolecule is streptavidin or avidin.

In some preferred embodiments, the cap-dependent subtraction methodfurther comprises the step of: (v) washing the surface to which thecaptured modified-nucleotide-capped RNA derived from the first sample isbound under conditions wherein the RNA that was digested by RNase H isremoved and wherein other nucleic acids, including the first-strand cDNAprepared from the RNA derived from the second sample is removed. Thus,in some embodiments of the kit for CDS, the kit additionally comprises asolution for washing the surface to which the modified-nucleotide-cappedRNA is bound.

In some preferred embodiments, the cap-dependent subtraction methodfurther comprises the step of: (vi) contacting the capturedmodified-nucleotide-capped RNA derived from the first sample thatremains bound to the surface with a protein or biochemical reagent underconditions wherein the triphosphate between the modified cap nucleotideand the 5′-penultimate nucleotide of the modified-nucleotide-capped RNAis cleaved, thereby de-capping the modified-nucleotide-capped RNAderived from the first sample and releasing the de-capped RNA derivedfrom the first sample. Thus, some embodiments of the kit for CDSadditionally comprise a pyrophosphatase or decapping enzyme. In someembodiments, the pyrophosphatase or decapping enzyme is selected fromamong tobacco acid pyrophosphatase and yeast or human decapping enzyme.

In the embodiments of cap-dependent subtraction, the de-capped RNAderived from the first sample that is released comprises those RNAmolecules in the first sample which are not present in the second sampleor which, if they are present in the second sample, are present insubstantially lower quantity than in the first sample. In someembodiments, the first-strand cDNA prepared using RNA derived from thesecond sample is prepared from RNA amplified in an RNA amplificationreaction, whereby the amount of first-strand cDNA that is preparedtherefrom is in significant excess over the amount of the capturedmodified-nucleotide-capped RNA derived from the first sample, therebyincreasing the probability that the captured modified-nucleotide-cappedRNA derived from the first sample that is not digested by RNase H duringthe method is present only in the first sample or, is present insignificantly larger amount in the first sample. In some embodimentswherein the method for cap-dependent subtraction (CDS) is used, theuncapped RNA derived from the first sample or first cell and/or the RNAderived from the second sample or second cell that used to synthesizethe first-strand cDNA for the CDS method are synthesized using an invitro RNA amplification reaction. In preferred embodiments, the in vitroRNA amplification reaction is a sense RNA amplification reaction thatsynthesizes uncapped RNA products comprising amplified sense RNA, alsocalled “sense RNA”, wherein the sequence is not different from thesequence of the RNA (e.g., from a cell, tissue, organ, or otherbiological sample) that is amplified using said sense RNA amplificationmethod. In some preferred embodiments, the sense RNA amplificationmethod is performed as described in U.S. Patent Application No.20050153333 of Sooknanan; U.S. Patent Application No. 20030186237 ofGinsberg, Stephen; U.S. Patent Application No. 20040197802 of Dahl andJendrisak; and U.S. Patent Application No. 20040171041 of Dahl et al.;or in Ozawa, T et al. (Biotechniques 40: 469-478, 2006). In someembodiments of a kit of the invention, the kit additionally comprisesone or more enzymes or reagents for performing a sense RNA amplificationreaction, including, but not limited to, a sense RNA amplificationreaction as described above.

Thus, the cap-dependent subtraction (CDS) method provides a way toobtain a population of RNA molecules that is specific for the type ofcell (i.e., “type-specific”) or for the condition(s) to which it issubjected (i.e., “condition-specific”). This population of RNA molecules(sometimes referred to “subtracted RNA”) is useful for further analysisor use. By way of example, but without limitation, the subtracted RNAcan be identified (e.g. by analysis on an Affymetrix, Agilent, Illumina,or NimbleGen Systems microarray chip). If the subtracted RNA is from acell with a condition, such as a cancer cell, or a cell from anotherorganic disease, or a cell that is infected with a bacterial,mycoplasmal, fungal, or viral pathogen, it comprises a population ofpotential pharmaceutical drug targets, which, if further validated, canbe used to develop pharmaceuticals to relieve symptoms or potentiallycure the disease. Of course, a validated condition-specific target canalso be used to develop human or animal diagnostic tests, assays andkits. The subtracted RNA is also useful for research purposes. Forexample, in one embodiment, subtracted RNA from a cancer stem cell iscompared with subtracted RNA from normal cells of the same type and/orother cancer cells which are not stem cells from the cancer lesion inorder to understand the progression of the cancer and develop therapiesand treatments. In still another embodiment, the subtracted RNA is usedfor synthesis of capped and polyadenylated RNA, which is further usedfor making an RNA-loaded antigen-presenting cell (APC) for use as avaccine to prevent or treat a disease (as discussed elsewhere, herein);for example, in some embodiments, subtracted RNA from the cancer stemcell from a tumor from a patient is used to make capped andpolyadenylated RNA for use in transforming a dendritic cell preparedfrom the same patient, wherein the dendritic cell that is loaded withthe tumor-specific RNA presents tumor-specific antigens. The tumorantigen-presenting dendritic cells are used to make a vaccine to attemptto induce a cell-mediated immune response in the patient. In stillanother embodiment, the tumor antigen-presenting dendritic cells areused to make cytotoxic T-lymphocytes (CTLs) in culture, and the CTLs areused to make a vaccine to treat the patient.

In some preferred embodiments, the protein or biochemical reagent thatcleaves the triphosphate is a pyrophosphatase or decapping enzyme. Insome preferred embodiments, the protein or biochemical reagent thatcleaves the triphosphate is selected from the group consisting oftobacco acid pyrophosphatase, Saccharomyces cerevisiae decapping enzyme,and human decapping enzyme. Thus, the method provides a substantiallypurified de-capped RNA from the captured modified-nucleotide-capped RNAthat is bound to the surface. The substantially purified de-capped RNAis useful for additional analysis and use. Thus, in some embodiments ofthe kit for CDC, the kit additionally comprises: (vi) a protein orbiochemical reagent that is capable of specifically cleaving thetriphosphate in the modified-nucleotide-capped RNA between the modifiedcap nucleotide and the 5′-penultimate nucleotide of themodified-nucleotide-capped RNA without cleaving other positions in themodified-nucleotide-capped RNA. In some preferred embodiments of thekit, the protein or biochemical reagent that is capable of specificallycleaving the triphosphate selected from the group consisting of tobaccoacid pyrophosphatase, Saccharomyces cerevisiae decapping enzyme, andhuman decapping enzyme.

The methods for cap-dependent capture (CDC) and for cap-dependentsubtraction (CDS) are very amenable to automation. Thus, in someembodiments, one or both of these methods is automated using a robotthat is capable of robotic aliquoting, mixing, washing,temperature-controlled incubation, and liquid collection and transfer.

C. Detailed Description of Cap-Dependent Capture, Including Use forCapture, Isolation, and Purification of Prokaryotic Bacterial mRNA andProducts from Transcription and RNA Amplification Reactions

In some embodiments, wherein the method for cap-dependent capture (CDC)is used, the uncapped RNA comprises primary RNA transcripts from aprokaryotic or eukaryotic biological sample. In some preferredembodiments wherein the method for cap-dependent capture (CDC) is used,the uncapped RNA comprises prokaryotic (e.g., bacterial or mycoplasmal)primary RNA transcripts from a biological sample. In some embodimentswherein the CDC method is used to capture prokaryotic primary RNAtranscripts, the sample contains both prokaryotic and eukaryotic mRNA.In some embodiments, the sample contains both one or more prokaryoticmRNA molecules and one or more eukaryotic mRNA molecules. The CDC methodcan be used to capture the prokaryotic mRNA in the sample withoutcapturing the eukaryotic mRNA because the 5′-end of mRNA from abacterial prokaryote has a 5′-triphosphate and is usually not capped,whereas the 5′-end of mRNA from a eukaryote is usually capped. Thus, themethod can be used to capture prokaryotic (e.g., bacterial ormycoplasmal) mRNA in a sample that also contains capped eukaryotic mRNA.Thus, one embodiment of the invention is a method for selectivelycapturing or isolating prokaryotic bacterial mRNA, the method comprising(i) providing uncapped RNA comprising prokaryotic bacterial mRNA, acapping enzyme system, a modified cap nucleotide, and a surface, towhich an affinity-tag-binding molecule is attached; (ii) contacting theuncapped RNA with the capping enzyme system and the modified capnucleotide under conditions wherein the prokaryotic bacterial mRNA iscapped and modified-nucleotide-capped RNA, which comprises theprokaryotic bacterial mRNA, is synthesized; and (iii) bindingmodified-nucleotide-capped RNA to the surface, whereby themodified-nucleotide-capped RNA, which comprises the prokaryoticbacterial mRNA, is captured. In some embodiments of this method, themodified-nucleotide-capped RNA synthesized has an affinity tag. In someembodiments of the method, the modified-nucleotide-capped RNAsynthesized does not have an affinity tag and the method additionallyprovides an affinity tag reagent and the method further comprises thestep of (iv) contacting the modified-nucleotide-capped RNA with theaffinity tag reagent under conditions wherein the affinity tag is joinedto the modified cap nucleotide therein, and modified-nucleotide-cappedRNA that has an affinity tag is obtained. In some preferred embodiments,the method further comprises the step of: (v) contacting themodified-nucleotide-capped RNA that has the affinity tag with thesurface to which the affinity-tag-binding molecule is attached underconditions wherein the modified-nucleotide-capped RNA is captured.

In some embodiments, the affinity-tag-binding molecule is attached to asurface selected from among: a glass slide, a dipstick, an array ormicroarray chip, a magnetic bead, a gold particle, a quantum dot, amicrochannel in glass, silica or another material, a well of a tube, awell of a microtiter plate, or another surface known in the art that iscapable of being used for attaching an appropriate affinity-tag-bindingmolecule. Many methods are known in the art for binding anaffinity-tag-binding molecule to a surface, which can be used for thepresent invention. In some preferred embodiments of the above methods,the affinity tag is biotin and the affinity-tag-binding molecule isstreptavidin or avidin. In some embodiments wherein an affinity tagreagent used, it is a biotinylation reagent. Thus, in some embodimentswherein the modified-nucleotide-capped RNA comprising biotin is bound tothe surface to which streptavidin or avidin is attached, the prokaryoticmRNA is bound or captured, and thereby isolated from the mixture ofnucleic acids and other molecules in the sample. The method can be usedfor a variety of applications. For example, but without limitation, insome embodiments, the prokaryotic mRNA is mRNA of a bacterial pathogenthat is in the presence of mRNA of a eukaryotic host cell, and themethod enables capture, isolation and purification of the mRNA of thebacterial pathogen, e.g., for further identification, analysis, and use.Thus, in some embodiments, the method for capturing the pathogenicbacterial mRNA can comprise a part of a molecular diagnostic method,assay or test; for example, the pathogenic bacterial mRNA on thesurface, for example consisting of a dipstick, an array or microarraychip, magnetic beads, the inside of a tube, or the wells of a microtiterplate, can be annealed or hybridized to a labeled nucleic acid probethat is specific for the pathogen, thereby enabling detection andidentification of the presence of the pathogen. In some embodiments, themolecular diagnostic method, assay or test can be configured to permitquantification of the amount of the pathogenic mRNA present in thesample, which can thereby indicate the quantity of the pathogen bacteriapresent in the sample. Without limitation, in still other embodiments,the captured or isolated prokaryotic mRNA that is released from thesurface is amplified using a method known in the art for synthesizingamplified sense RNA having the same sequence as the prokaryotic mRNA,and this amplified sense RNA is contacted with a capping enzyme systemand a cap nucleotide under conditions wherein it is capped, andcontacted with a poly(A) polymerase and ATP under conditions wherein apoly(A) tail is added to the 3′-terminus, thereby obtaining 5′-cappedamplified sense RNA having a 3′-poly(A) tail; in some embodiments thecap nucleotide is a modified cap nucleotide; in other embodiments, theamplified sense RNA is capped using a dinucleotide cap analog in an invitro transcription reaction that is part of the sense RNA amplificationmethod. In some preferred embodiments, the invention further comprisesusing the 5′-capped amplified sense RNA having a 3′-poly(A) tail to makea vaccine to prevent or treat a condition using methods describedelsewhere herein. For example, but without limitation, in one preferredembodiment, the 5′-capped amplified sense RNA having a 3′-poly(A) tailthat is synthesized using prokaryotic mRNA from a pathogenic bacteriumor from a sample from a human or animal patient infected with thebacterium is used for transforming an antigen-presenting cell (APC),such as a dendritic cell, a macrophage, an epithelial cell, or anartificial APC, and using said transformed APC to make the vaccine.Alternatively, in some embodiments, said transformed APC is used asdescribed elsewhere herein to make a cytotoxic T-lymphocyte (CTL) foruse as a vaccine to prevent or treat a human or animal patient with thepathogen-infected condition.

As a further example wherein the cap-dependent capture (CDC) method canbe used, in some embodiments, the uncapped RNA in the sample comprisesprokaryotic mRNA from a plant root nodule that is infected with asymbiotic nitrogen-fixing Rhizobium bacterium, which prokaryotic mRNA isalso in the presence of the eukaryotic plant mRNA. Since the planteukaryotic mRNA is already capped, the Rhizobial mRNA can be selectivelycaptured using the CDC method.

Although most eukaryotic mRNA has a poly(A) tail that can be used forselective synthesis of cDNA from the eukaryotic mRNA, which is a step inmany RNA amplification methods (e.g., which are used to make labeledtarget RNA for use in gene expression analysis (e.g., using a microarrayfrom Affymetrix, Agilent, Illumina, or NimbleGen Systems), mostprokaryotic bacterial mRNA does not have a poly(A) tail that is capableof being used for such purposes. Thus, the present invention provides anovel method for selective capture and isolation of prokaryotic (e.g.,bacterial) mRNA even in the presence of eukaryotic mRNA. Thus, the CDCmethod, when used in combination with methods known in the art, isenabling and extremely useful for analyzing gene expression by both theprokaryote and by the eukaryote using the same cell, tissue, organ, orother biological sample, thereby providing much better understanding ofwhat is occurring when during the infection process. Thus, thecap-dependent capture (CDC) method has important applications foragriculture, human and animal medicine, and biomedical research.

In some embodiments wherein the method for cap-dependent capture (CDC)is used, the uncapped RNA comprises primary RNA transcripts or RNAhaving a 5′-diphosphate that is used for the method is the product of anin vitro transcription reaction using an RNA polymerase. In someembodiments, the RNA polymerase is a wild-type or mutant T7-type RNApolymerase. In some embodiments the T7-type RNA polymerase is selectedfrom the group consisting of T7 RNA polymerase, T3 RNA polymerase, andSP6 RNA polymerase.

In some embodiments wherein the method for cap-dependent capture (CDC)is used, the uncapped RNA that is used for the method comprises primaryRNA transcripts or RNA having a 5′-diphosphate that are products of anin vitro RNA amplification reaction. In some preferred embodiments, thein vitro RNA amplification reaction is a sense RNA amplificationreaction that synthesizes uncapped RNA products comprising amplifiedsense RNA, also called “sense RNA”, wherein the sequence is notdifferent from the sequence of the RNA (e.g., from a cell, tissue,organ, or other biological sample) that is amplified using said senseRNA amplification method. Thus, in some preferred embodiments, theuncapped RNA comprising primary RNA transcripts or RNA having a5′-diphosphate that is used for the method is a product of a sense RNAamplification reaction, such as, but not limited to, a method thatsynthesizes sense RNA as described in U.S. Patent Application No.20050153333 of Sooknanan; U.S. Patent Application No. 20030186237 ofGinsberg, Stephen; U.S. Patent Application No. 20040197802 of Dahl andJendrisak; and U.S. Patent Application No. 20040171041 of Dahl et al.;or in Ozawa, T et al. (Biotechniques 40: 469-478, 2006). In someembodiments of a kit of the invention, the kit additionally comprisesone or more enzymes or reagents for performing a sense RNA amplificationreaction, including, but not limited to, a sense RNA amplificationreaction as described above.

However, in some embodiments, the uncapped RNA that is used for the CDCmethod is a product of an anti-sense RNA amplification reaction, suchas, but not limited to an anti-sense RNA amplification reactiondescribed in Murakawa et al., DNA 7:287-295, 1988; Phillips andEberwine, Methods in Enzymol. Suppl. 10:283-288, 1996; Ginsberg et al.,Ann. Neurol. 45:174-181, 1999; Ginsberg et al., Ann. Neurol. 48:77-87,2000; VanGelder et al. Proc. Natl. Acad. Sci. USA 87:1663-1667, 1990;Eberwine et al., Proc. Natl. Acad. Sci. USA 89:3010-3014, 1992; U.S.Pat. Nos. 5,021,335; 5,168,038; 5,545,522; 5,514,545; 5,716,785;5,891,636; 5,958,688; 6,291,170; and PCT Patent Applications WO 00/75356and WO 02/065093. In some embodiments of a kit of the invention, the kitadditionally comprises one or more enzymes or reagents for performing ananti-sense RNA amplification reaction, including, but not limited to, ananti-sense RNA amplification reaction as described above.

The RNA that is amplified in the RNA amplification reaction (e.g., ineither a sense RNA amplification reaction or an anti-sense RNAamplification reaction) in order to obtain uncapped RNA for use in thecap-dependent capture (CDC) method can be from one of a variety ofsources. In some embodiments, the RNA that is amplified is from abiological sample, such as a cell, tissue, or organ of a human, plant,animal, bacterium, virus, or fungus, or from a preparation of RNAprepared therefrom using an RNA extraction or purification method or kitknown in the art. In other embodiments, the RNA that is amplified toobtain the uncapped RNA for subsequent use in the CDC method isde-capped RNA obtained from a prior round of the CDC method, wherein thede-capped RNA comprises RNA that is released from the prior round of CDCby contacting the captured modified-nucleotide-capped RNA with apyrophosphatase. In still other embodiments, the RNA that is amplifiedto obtain the uncapped RNA for subsequent use in the CDC method isde-capped RNA obtained from a prior round of the cap-dependentsubtraction (“CDS”) method described elsewhere herein; in thisembodiment, the RNA that is amplified comprises “type-specific” or“condition-specific” RNA, from which RNA that is in common with RNA inanother sample has been subtracted.

Based on the above description, it will be clear that the methods of theinvention comprising contacting an uncapped RNA with a capping enzymesystem and a modified cap nucleotide whereby modified-nucleotide-cappedRNA is synthesized (including the CDC method) can be used with anyuncapped RNA comprising primary RNA transcripts or RNA having a5′-diphosphate, whether the uncapped RNA is obtained from an in vivo orbiological source, or from an in vitro source. In fact, with respect toRNA obtained in vitro, each round of in vitro transcription or RNAamplification provides new uncapped RNA comprising primary RNAtranscripts or RNA having a 5′-diphosphate which can be capped with amodified cap nucleotide using the method of the invention. Theparticular modified cap nucleotide can be the same for capping RNA fromeach round of in vitro transcription or a different modified capnucleotide can be used for capping the uncapped RNA obtained from eachround. By way of example, but without limitation, in some embodiments,2′-amino-2′-dGTP is used as the modified cap nucleotide for cappingprimary mRNA transcripts from a first round of in vitro transcription orRNA amplification, permitting isolation of themodified-nucleotide-capped RNA obtained using the CDC method, and thenanother modified cap nucleotide (e.g., O⁶-Me-GTP) is used for cappingprimary mRNA transcripts from a second round of in vitro transcriptionor RNA amplification, permitting higher efficiency translation of theRNA into protein in a eukaryotic cell (e.g., preferably after alsopolyadenylating the modified-nucleotide-capped RNA using poly(A)polymerase).

In some embodiments of the method in which prokaryotic bacterial mRNA iscaptured, isolated, purified and released by de-capping using the CDCmethods of the invention, the method further comprises tailing theprokaryotic mRNA, such as by contacting the mRNA with poly(A) polymeraseand ATP under conditions wherein a poly(A) tail is added to the3′-termini. In some embodiments of the method in which prokaryotic mRNAis captured, isolated, purified and released by de-capping using themethods of the invention, the method further comprises adding a terminalsequence tag to the 3′ termini of the released mRNA. In otherembodiments, the method further comprises contacting the released mRNAwith an RNA-dependent DNA polymerase and a primer under conditionswherein first-strand cDNA is synthesized and then, a tag is added to the3′ termini of the first-strand cDNA using the terminal tagging methoddescribed by Sooknanan in U.S. Patent Application Nos. 20050153333. Instill other embodiments of the method in which prokaryotic mRNA iscaptured, isolated, purified and released by de-capping using the methodof the invention, the method further comprises synthesizing first-strandcDNA by reverse transcription of the mRNA and tailing the first-strandcDNA by contacting the cDNA with a terminal transferase, such as calfthymus terminal deoxynucleotidyl transferase, in the presence of atleast one deoxyribonucleoside-5′-triphosphate under terminal transferasereaction conditions. In some embodiments of the method in which aterminal sequence tag and/or a tail is added to the 3′ termini ofprokaryotic mRNA and/or cDNA derived by reverse transcription of themRNA, the method further comprises amplifying the prokaryotic mRNA usingan RNA amplification reaction. In preferred embodiments, the RNAamplification reaction synthesizes sense RNA. In other embodiments, theRNA amplification reaction synthesizes anti-sense RNA.

In some embodiments of the kit or method wherein the modified capnucleotide has a 2′ or 3′ substituent consisting of an amino or an azidogroup or a thiol substituent on the 6-position of the guanine base, thekit or method additionally provides: an affinity tag reagent; and anaffinity-tag-binding molecule, which is either free or attached to asurface; and the method additionally comprises the steps of: (i)contacting the modified-nucleotide-capped RNA with the affinity tagreagent under conditions wherein the affinity tag is chemically joinedto the modified cap nucleotide of the modified-nucleotide-capped RNA,whereby modified-nucleotide-capped RNA having an affinity tag isobtained; and (ii) contacting the modified-nucleotide-capped RNA havingthe affinity tag with the affinity-tag-binding molecule under conditionswherein the modified-nucleotide-capped RNA having the affinity tag isbound, and the modified-nucleotide-capped RNA is captured, isolated orlabeled. In some embodiments of the kit or method wherein the affinitytag comprises biotin, the affinity-tag-binding molecule is streptavidinor avidin, either free or attached to a surface and/or attached toanother molecule, including, but not limited to a fluorescent or otherdetectable label.

In some embodiments of the kit or method, the affinity tag having thereactive moiety is a biotinylation reagent. Any biotinylation reagentknown in the art that reacts with the amino, azido, or thiol group onthe 2′- or 3′-position of the sugar or the thiol group on the 6 positionof the guanine base of the respective modified cap nucleotide can beused in the methods and kits of the invention. Without limitation, somebiotinylation reagents that can be used are described in “Avidin-BiotinChemistry: A Handbook”, by D. Savage et al., Pierce Chemical Company,1992 and in “Handbook of Fluorescent Probes and Research Products”,Ninth Edition, by R. P. Hoagland, Molecular Probes, Inc. Thus, withoutlimitation, in some embodiments of the kit or method wherein themodified cap nucleotide comprises a 2′- or 3′ thiol group, or6-thioguanine, the biotinylation reagent comprises a thiol-reactiveiodoacetamidyl-, iodoacetyl-, or maleimidyl-moiety. In some otherembodiments of the kit or method wherein the modified cap nucleotidecomprises a 2′- or 3′-amino or azido group, the affinity tag reagent iscomprises a 6-[(+)-biotinamidocaproyl]-group or a6-[(+)-biotinamidocaproylamido]-caproyl-group that has a reactive moietycomprising an N-hydroxysuccinimidyl (i.e., “NHS”) ester that is capableof reacting with the amino group, or a reactive moiety comprising analkynyl group that is capable of reacting with the azido group. Forexample, in some embodiments of the kit or method wherein the 2′ or 3′substituent of the modified cap nucleotide is an amino group, theaffinity tag reagent is biotin-X-X-NHS (also called biotin-LC-LC-NHS or6-[(+)-biotinamidocaproylamido]-caproic acid N-hydroxysuccinimide ester)or biotin-X-NHS (also called biotin-LC-NHS). In other embodiments, adifferent biotinylation known in the art with a shorter or longer sidechain and/or with a different reactive moiety is used. In someembodiments of the kit or method wherein the 2′ or 3′ substituent of themodified cap nucleotide is an azido group, the reactive moiety of theaffinity tag is a reactive alkynyl group which is capable of joining theaffinity binding molecule to the azido group via a 1,3-dipolarcycloaddition; in embodiments of the method, the affinity tag is joinedto the modified cap nucleotide of the modified-nucleotide-capped RNA viaa 1,2,3-triazole moiety. Affinity tag reagents having a reactive moietyconsisting of an alkynyl group which can be used for the kits andmethods of the present invention are known in the art (e.g., seeBreinbauer, R and Kohn, M, ChemBioChem 4: 1147-1149, 2003), and thosewith knowledge in the art will understand how to obtain an affinity tagreagent having a reactive alkynyl group for other applications usingsuch described methods. By way of example, but without limitation, theinvention comprises a method for obtaining an affinity tag reagenthaving a reactive alkynyl group (e.g., a biotinylation reagent having areactive alkynyl group), the method comprising reacting a compoundhaving an affinity tag reagent having an amine-reactive moiety (e.g.,biotin-X-X-NHS) with a molecule having a primary amine and an alkynylgroup, thereby obtaining the affinity tag reagent having a reactivealkynyl group (e.g., biotin-X-X-NH-alkyne). Similarly, bioconjugationtechniques for using such affinity tag reagents having a reactivealkynyl group (e.g., for bioconjugation to a modified-nucleotide-cappedRNA having a modified cap nucleotide with a 2′ or 3′ azido group) areknown in the art (e.g., see Breinbauer, R and Kohn, M, ChemBioChem 4:1147-1149, 2003), which can be used in the kits and methods of theinvention.

Thus, some embodiments of the invention comprise a method comprising:(i) contacting an uncapped RNA comprising primary RNA transcripts or RNAhaving a 5′-phosphate with a capping enzyme and a modified capnucleotide containing an amino, azido or thiol group under conditionswherein a modified-nucleotide-capped RNA comprising a modified capnucleotide having a reactive amino, azido or thiol group is synthesized.In some embodiments, the method further comprises one or more of thefollowing steps: (ii) contacting the modified-nucleotide-capped RNA withan affinity tag reagent that is capable of chemically joining theaffinity tag to the respective amino, azido or thiol group of themodified-nucleotide-capped RNA under conditions whereinmodified-nucleotide-capped RNA having the affinity tag is synthesized;(iii) contacting the modified-nucleotide-capped RNA having the affinitytag to a surface to which an affinity-tag-binding molecule that iscapable of binding the affinity tag, thereby binding and capturing orisolating the modified-nucleotide-capped RNA having the affinity tag;(iv) washing the surface to which the modified-nucleotide-capped RNAhaving the affinity tag is bound, thereby removing contaminants,including nucleic acids, that are not bound and purifying themodified-nucleotide-capped RNA having the affinity tag which is bound tothe surface; and (v) contacting the surface to which themodified-nucleotide-capped RNA having the affinity tag is bound with anenzyme or reagent that cleaves the RNA of the modified-nucleotide-cappedRNA from the bound modified cap nucleotide having the affinity tag,thereby releasing the purified RNA for further analysis and use.

Thus in some specific embodiments, the invention provides a kit forcapturing or isolating uncapped RNA comprising primary RNA transcriptsor RNA having a 5′-diphosphate, the kit comprising a capping enzymesystem and a modified cap nucleotide selected from among:2′-amino-2′-dGTP; 3′-amino-3′-dGTP; 2′-amino-2′-ddGTP;3′-amino-3′-ddGTP; 2′-azido-2′-dGTP; 3′-azido-3′-dGTP;2′-azido-2′-ddGTP; 3′-azido-3′-ddGTP; 2′-mercapto-2′-dGTP (i.e.2′-SH-2′-dGTP); 3′-mercapto-3′-dGTP; 2′-mercapto-2′,3′-dGTP;3′-mercapto-2′,3′-ddGTP; 6-thio-GTP (i.e., 6-mercapto-GTP);6-thio-2′-dGTP; and 6-thio-3′-dGTP. The invention also provides a methodfor capturing and isolating uncapped RNA comprising primary RNAtranscripts or RNA having a 5′-diphosphate, the method comprising: (i)providing an uncapped RNA comprising primary RNA transcripts or RNAhaving a 5′-diphosphate; a capping enzyme system; and a modified capnucleotide selected from among 2′-amino-2′-dGTP, 3′-amino-3′-dGTP,2′-amino-2′-ddGTP, 3′-amino-3′-ddGTP, 2′-azido-2′-dGTP,3′-azido-3′-dGTP, 2′-azido-2′-ddGTP, 3′-azido-3′-ddGTP,2′-mercapto-2′-dGTP; 3′-mercapto-3′-dGTP; 2′-mercapto-2′,3′-dGTP;3′-mercapto-2′,3′-ddGTP; 6-thio-GTP, 6-thio-2′-dGTP and 6-thio-3′-dGTP;(ii) contacting the uncapped RNA with the capping enzyme system and themodified cap nucleotide under conditions wherein themodified-nucleotide-capped RNA is synthesized; (iii) contacting themodified-nucleotide-capped RNA with a biotinylation reagent (e.g.,respectively, biotin-NHS ester or biotin-X-NHS ester or biotin-X-X-NHSester or biotin-X-X-sulfo-NHS ester; biotin-X-X-propyne; andbiotin-maleimide or biotin iodoacetamide) under conditions wherein therespective amino, azido, or thiol group is biotinylated and abiotinylated modified-nucleotide-capped RNA is obtained; (iv) contactingthe biotinylated modified-nucleotide-capped RNA with streptavidin oravidin (e.g., that is attached to a surface) under conditions whereinthe biotinylated modified-nucleotide-capped RNA is bound (e.g., to thesurface); and (v) separating the bound biotinylatedmodified-nucleotide-capped RNA from unbound molecules (e.g., bywashing). In some embodiments, the method further comprises the stepsof: (vi) contacting the bound biotinylated modified-nucleotide-cappedRNA with a pyrophosphatase (e.g., tobacco acid pyrophosphatase) underpyrophosphatase reaction conditions or a decapping enzyme (e.g., yeastor human decapping enzyme) under decapping enzyme reaction conditions,whereby the biotinylated modified-nucleotide-capped RNA that is bound(e.g., to a surface) is de-capped and the de-capped RNA is released; and(vii) obtaining the de-capped RNA that is released. Thus, in someembodiments, the kit further comprises a pyrophosphatase (e.g., tobaccoacid pyrophosphatase) or a decapping enzyme (e.g., yeast or humandecapping enzyme).

In some other specific embodiments, modified-nucleotide-capped RNA,prepared from a uncapped RNA comprising prokaryotic mRNA using avaccinia capping enzyme system and 2′-amino-dGTP, is reacted withbiotin-X-X-NHS ester (EPICENTRE Biotechnologies, Madison, Wis., USA) inthe presence of 1-methyl-imidazole as a catalyst. The 2′-amino group ofthe modified cap nucleotide of the modified-nucleotide-capped RNA isthereby derivatized with a biotin affinity tag molecule, which is usedto capture the biotinylated modified-nucleotide-capped RNA usingstreptavidin that is attached to a surface. In some embodiments, thesurface is a magnetic bead, a dipstick, a membrane, the surface of thewells of a microtiter plate, an array or microarray slide of chip, oranother solid or porous surface. In some embodiments, the biotinylatedmodified-nucleotide-capped RNA that is so captured is released byincubating with tobacco acid pyrophosphatase in 1× reaction buffer asdescribed by the supplier (EPICENTRE Biotechnologies, Madison, Wis.,USA), or by incubating with yeast or human decapping enzyme usingconditions known in the art. The biotinylated modified-nucleotide-cappedRNA can also be labeled by binding to streptavidin or avidin that iscovalently attached to a detectable molecule, including, without limit,a visible, fluorescent, luminescent, or infrared fluorescent molecule,such as, but not limited to, phycoerythrin or another phycobiliprotein,fluorescein, rhodamine, Cy3, Cy5, or an Alexa dye, or another dye, or tostreptavidin or avidin that is covalently attached to an enzyme that isdetectable using a substrate, such as, but not limited to, a substratethat results in a visible, fluorescent, luminescent, or infraredfluorescent signal under enzymatic reaction conditions. The binding ofthe labeled streptavidin or avidin affinity-tag-binding molecule to thebiotinylated modified-nucleotide-capped RNA provides a detection methodfor uses such as, but not limited to, detecting a nucleic acid sequenceas part of a diagnostic assay or for assay of gene expression on anarray or microarray.

In other specific embodiments of the method in which the modified capnucleotide is a 2′-amino- or 3′-amino-modifieddeoxyguanosine-5′-triphosphate (i.e., 2′-amino-2′-dGTP or3′-amino-3′-dGTP), the method further comprises the step of reacting themodified-nucleotide-capped RNA with a reactive detectable dye underconditions wherein the amino group is labeled with the dye underconditions wherein detectable modified-nucleotide-capped RNA isobtained. Thus, in some embodiments of the method in which the modifiedcap nucleotide is a 2′-amino- or 3′-amino-modifieddeoxyguanosine-5′-triphosphate (i.e., 2′-amino-2′-dGTP or3′-amino-3′-dGTP), the method further comprises reacting themodified-nucleotide-capped RNA with a reactive detectable dye, such as,but not limited to, a fluorescent dye, a luminescent dye, a visible dye,or infrared fluorescent dye, such as selected from the group consistingof a N-hydroxysuccinimidyl ester of a Cy dye, a rhodamine dye, afluorescein dye, and another dye that is known in the art. Thus, themethod of the present invention also provides a way to label uncappedRNA comprising primary RNA transcripts or RNA having a 5′-diphosphatewith a detectable dye.

In embodiments of the method in which the modified cap nucleotide is a2′-azido- or 3′-azido-modified deoxyguanosine-5′-triphosphate (i.e.,2′-azido-2′-dGTP or 3′-azido-3′-dGTP), the method further comprisesreacting the modified-nucleotide-capped RNA with a compound containingan alkynyl or acetylene moiety under conditions wherein the alkynylmoiety reacts with the azido group of the modified capped nucleotideunder conditions wherein a derivatized modified-nucleotide-capped RNA isobtained. The reagent can provide a moiety for binding and capturing themodified-nucleotide-capped RNA or for labeling themodified-nucleotide-capped RNA with a detectable dye or it can provideanother functionality. Detectable dyes having a reactive alkynyl group,such as, but not limited to, fluorescein dyes (Wang, Q et al., J. AmChem. Soc. 125: 3192-3193, 2003) and rhodamine dyes (Speers, A E et al.,J. Am Chem. Soc. 124: 4686-4687, 2003) are known in the art, which canbe used in embodiments of kits and methods of the invention. In otherembodiments, a detectable molecule having an alkynyl group is made usingconventional synthesis techniques known in the art (e.g., seeBreinbauer, R and Kohn, M, ChemBioChem 4: 1147-1149, 2003; Campbell, D Aand Szardenings, A K, Curr. Opin. Chem. Biol. 7: 296-303, 2003), and theinvention includes use of any such detectable molecules in the kits andmethods of the invention. The invention also includes the use oftechniques known in the art for coupling such detectable moleculeshaving a reactive alkynyl group, such as, but not limited tofluorescein, rhodamine, or other dyes having an alkynyl group, to anazido substituent (e.g., see Wang, Q et al., J. Am Chem. Soc. 125:3192-3193, 2003; Speers, A E et al., J. Am Chem. Soc. 124: 4686-4687,2003; Breinbauer, R and Kohn, M, ChemBioChem 4: 1147-1149, 2003;Campbell, D A and Szardenings, A K, Curr. Opin. Chem. Biol. 7: 296-303,2003), wherein the azido substituent is the 2′ or 3′ azido substituentof a modified cap nucleotide in a modified-nucleotide-capped RNA.

By way of example, but without limitation, in one embodiment, onehundred molar equivalents of modified-nucleotide-capped RNA, preparedfrom uncapped RNA comprising primary RNA transcripts or RNA having a5′-diphosphate using a capping enzyme system and 2′-azido-dGTP as amodified cap nucleotide, is reacted with 117 equivalents of an alkynylflurorescein in the presence of 50 equivalents of CuSO₄ and 100equivalents of tris(carboxethyl)phosphine (TCEP) as described by Wang, Qet al. (J. Am. Chem. Soc. 125: 3192, 2003). The 2′-azido group of themodified cap nucleotide of the modified-nucleotide-capped RNA is therebylabeled with fluorescein dye. In another embodiment, one molarequivalent of modified-nucleotide-capped RNA, prepared from uncapped RNAderived from primary RNA using a capping enzyme system and 2′-azido-dGTPas a modified cap nucleotide, is reacted with 1.2 equivalents of analkynyl rhodamine in the presence of 0.5 equivalent of CuSO₄ and oneequivalent of TCEP as described by Speers, A E et al. (J. Am. Chem. Soc.124: 4686, 2003). The 2′-azido group of the modified cap nucleotide ofthe modified-nucleotide-capped RNA is thereby labeled with rhodaminedye. The flourescein- or rhodamine-labeled modified-nucleotide-cappedRNA provides a detection method for uses such as, but not limited to,detecting a nucleic acid sequence as part of a diagnostic assay or forassay of gene expression on an array or microarray, or for any of theother many applications known in the art where a labeled nucleic isuseful.

In some embodiments of the method wherein the modified cap nucleotidehas a 2′ or 3′ azido group, the modified-nucleotide-capped RNA consistsof one or more prokaryotic mRNA molecules, including, but not limitedto, all prokaryotic mRNA molecules in the sample. Since mRNA from aeukaryotic cell is already capped, prokaryotic mRNA in a sample thatalso contains eukaryotic mRNA is preferentially capped with a modifiedcap nucleotide by a capping enzyme system using a method and/or kit ofthe invention. In other embodiments, the modified-nucleotide-capped RNAconsists of one or more uncapped RNAs comprising primary RNA transcriptsor RNAs having a 5′-diphosphate from an in vitro transcription reaction,including one or more uncapped RNAs comprising primary RNA transcriptsor RNAs having a 5′-diphosphate obtained from an RNA amplificationreaction, such as an RNA amplification reaction described in thereferences cited above. In preferred embodiments the one or moreuncapped RNAs comprise sense RNA. In some embodiments of this aspect ofthe invention, wherein the reagent has an affinity tag or capture moiety(e.g., a biotinylation reagent), the method further comprises:contacting the modified-nucleotide-capped RNA with the reagent, whereinthe affinity tag is joined to the modified-nucleotide-capped RNA; andcontacting the affinity tag-joined modified-nucleotide-capped RNA withan affinity tag binding molecule that has affinity for and binds to thereagent and that is directly or indirectly, attached to a surface,whereby the affinity tag-joined modified-nucleotide-capped RNA isisolated. Thus, in one embodiment, the method is used to isolate one ormore prokaryotic mRNA molecules or one or more uncapped RNAs comprisingprimary RNA transcripts or RNAs having a 5′-diphosphate from an in vitrotranscription reaction. In some embodiments, the one or more prokaryoticmRNA molecules is in a sample comprising both prokaryotic and eukaryoticmRNA. For example, but without limitation, in some embodiments, theprokaryotic mRNA is mRNA of a bacterial pathogen that is in the presenceof mRNA of a eukaryotic host cell. In other embodiments, as a furtherexample, the mRNA is mRNA from a nitrogen-fixing Rhizobium bacteriumwhich is in the presence of mRNA from a legume root nodule. Sincebacterial mRNA generally does not have a poly(A), which is commonly usedto isolate eukaryotic mRNA, the method provides a novel method by whichto isolate prokaryotic mRNA. Once prokaryotic mRNA is obtained usingthis embodiment of the method of the invention, the invention furthercomprises tailing the prokaryotic mRNA, such as by contacting theisolated mRNA with poly(A) polymerase under reaction conditions or usingthe method described by Sooknanan in U.S. Patent Application Nos.20050153333, and further amplifying the prokaryotic mRNA using an RNAamplification method, such as a method cited herein above. In preferredembodiments, the RNA amplification reaction synthesizes sense RNA. Inother embodiments, the RNA amplification reaction synthesizes anti-senseRNA.

D. Detailed Description of the Cap-Dependent Subtraction Method toObtain Condition-Specific RNA

In some embodiments, the method further comprises the steps of:annealing to the modified-nucleotide-capped RNA that is bound by theaffinity-tag-binding molecule an excess of cDNA prepared from cellsdifferent from those used to obtain the modified-nucleotide-capped RNA;and treating the bound modified-nucleotide-capped RNA to which the cDNAis annealed with an RNase H, wherein modified-nucleotide-capped RNA towhich the cDNA is annealed is digested and modified-nucleotide-cappedRNA to which no cDNA is annealed is not digested and remains bound tothe affinity-tag-binding molecule, thereby subtracting themodified-nucleotide-capped RNA that is homologous to the cDNA. Thus, insome embodiments, the invention provides a method for subtracting fromthe population of all mRNA molecules derived from a first cell that isof a first type or that is under a first condition those mRNA moleculesthat are also present in a second cell of a second type or that areunder a second condition, thereby obtaining a population of mRNAmolecules that are present only in the first cell, but absent in thesecond cell, the method comprising: (i) obtainingmodified-nucleotide-capped RNA by contacting uncapped RNA comprisingprimary RNA transcripts or RNA having a 5′-diphosphate that is derivedfrom the first cell with a capping enzyme system and a modified capnucleotide consisting of a 2′ and/or 3′ deoxyguanosine-5′-triphosphatehaving a 2′ or 3′ amino, azido, or thiol (mercapto) substituent, underconditions wherein modified-nucleotide-capped RNA is synthesized fromthe mRNA from the first cell; (ii) reacting the 2′ or 3′ amino, azido,or thiol substituent with an affinity tag reagent that reacts therewith,thereby obtaining modified-nucleotide-capped RNA having the affinity tagon the amino, azido, or thiol substituent; (iii) preparing first-strandcDNA by reverse transcription of mRNA derived from the second cell usingan RNA-dependent DNA polymerase or reverse transcriptase and a primer,such as, but not limited to an oligo(dT) or a random sequence primer;(iv) annealing the first-strand cDNA prepared from mRNA from the secondcell to the modified-nucleotide-capped RNA from the first cell underhybridization conditions; (v) capturing the modified-nucleotide-cappedRNA having the affinity tag on a surface by binding the affinity tagwith an affinity-tag-binding molecule that is attached to a surface,thereby attaching the modified-nucleotide-capped RNA from the first cellto the surface; and (vi) treating the surface-attachedmodified-nucleotide-capped RNA from the first cell to which the cDNAfrom the second cell is annealed with RNase H, wherein the RNA to whichthe cDNA is annealed is digested, and subtractedmodified-nucleotide-capped RNA that is attached to the surface isobtained. In some embodiments of the method, step #(iv) is performedprior to step #(v), whereas in other embodiments step #(iv) is performedafter step #(v). In some embodiments of any of the above embodiments,the affinity tag having the reactive moiety is a biotinylation reagenthaving a reactive moiety consisting of an N-hydroxysuccinimidyl ester oran acylating or alkylating moiety for modified cap nucleosides having anamino substituent or an alkynyl moiety for modified cap nucleosideshaving an azido substituent; and the affinity-tag-binding molecule thatis attached to a surface is avidin or streptavidin. In some embodiments,the method further comprises the step of treating the subtractedmodified-nucleotide-capped RNA that is attached to the surface with apyrophosphatase or decapping enzyme, thereby releasing the subtractedRNA consisting of RNA from the first cell from which RNA that is incommon with RNA from the second cell has been removed or subtracted. Insome embodiments, the pyrophosphatase is tobacco acid pyrophosphatase.In some embodiments, the decapping enzyme is yeast or human decappingenzyme. The invention also comprises kits for performing the abovemethods. In one embodiment, the kit comprises: (a) a capping enzymesystem; (b) a modified cap nucleotide consisting of: (i) a 2′ or 3′deoxyguanosine-5′-triphosphate comprising a 2′ or 3′ amino, azido, orthiol substituent, (ii) 6-thioguanosine-5′-triphosphate (i.e., 6mercaptoguanosine-5′-triphosphate), or (iii) 6-thioguanine-2′- or3′-deoxynucleoside-5′-triphosphate; (c) an affinity tag reagent; and (d)an affinity-tag-binding molecule that is attached to a surface. In someembodiments of the kit, affinity tag reagent consists of a biotinaffinity tag having a reactive group consisting of, either, anN-hydroxysuccinimidyl ester or another acylating or alkylating moiety ifthe modified cap nucleotide has an amino substituent, or, an alkynylmoiety if the modified cap nucleotide has an azido substituent or, amaleimidyl moiety if the modified cap nucleotide has a thiol group; andthe affinity-tag-binding molecule that is attached to a surface consistsof avidin or streptavidin. In some of any of the above embodiments ofkits, the kit further comprises RNase H. In addition, any of the abovekits can also have one or more of the following reagents: a reversetranscriptase and/or a primer for preparing first-strand cDNA from mRNAfrom the second cell; and/or a hybridization solution for annealing thecDNA to the modified-nucleotide-capped RNA. The modified cap nucleotidein any of the above embodiments is selected from the group consisting of2′-amino-2′-deoxyguanosine-5′-triphosphate,3′-amino-3′-deoxyguanosine-5′-triphosphate,2′-azido-2′-deoxyguanosine-5′-triphosphate, and3′-azido-3′-deoxyguanosine-5′-triphosphate. The invention also comprisesthe subtracted RNA obtained using the above methods or kits.

The invention also provides kits and methods for labeling uncapped RNAcomprising primary RNA transcripts or RNA having a 5′-diphosphate,whether such transcripts are obtained from a biological sample, such asa cell or tissue, or are synthesized in vitro, such as in an in vitrotranscription or RNA amplification reaction, with a detectable molecule,such as, but not limited to, a visible, fluorescent, luminescent, orinfrared fluorescent dye, or an enzyme that is detectable using asubstrate, such as, but not limited to, a substrate that results in avisible, fluorescent, luminescent, or infrared fluorescent signal underenzymatic reaction conditions. For example, but without limitation, someembodiments provide a kit for indirect labeling comprising: a modifiedcap nucleotide selected from the group consisting of a guaninenucleoside-5′-triphosophate having 2′ or 3′ substituent consisting of anamino or an azido group; an affinity tag reagent; and anaffinity-tag-binding molecule that is labeled with the detectablemolecule. Some embodiments provide a method for indirect labeling of theuncapped RNA comprising primary RNA transcripts or RNA having a5′-diphosphate comprising: (i) providing an uncapped RNA comprisingprimary RNA transcripts or RNA having a 5′-diphosphate, a capping enzymesystem, a modified cap nucleotide having 2′ or 3′ substituent consistingof an amino or an azido group, an affinity tag reagent, and anaffinity-tag-binding molecule that is labeled with the detectablemolecule; (ii) contacting the uncapped RNA comprising primary RNAtranscripts or RNA having a 5′-diphosphate with the capping enzymesystem and the modified cap nucleotide under conditions wherein theuncapped RNA comprising primary RNA transcripts or RNA having a5′-diphosphate is capped and a modified-nucleotide-capped RNA issynthesized; (iii) contacting the modified-nucleotide-capped RNA withthe affinity tag having the reactive moiety under conditions whereinmodified-nucleotide-capped RNA having a modified cap nucleotide that isjoined to the affinity tag is obtained; and (iv) contacting the affinitytag that is joined to the modified cap nucleotide of themodified-nucleotide-capped RNA with the labeled affinity-tag-bindingmolecule under binding conditions, whereby themodified-nucleotide-capped RNA is labeled. The uncapped RNA comprisingprimary RNA transcripts or RNA having a 5′-diphosphate can be from abiological source or from an in vitro transcription reaction, includingan RNA amplification reaction. In some embodiments, the uncapped RNAcomprising primary RNA transcripts or RNA having a 5′-diphosphate is ina mixture that also includes other nucleic acids that are not uncappedRNA comprising primary RNA transcripts or RNA having a 5′-diphosphate.In some embodiments, the uncapped RNA comprising primary RNA transcriptsor RNA having a 5′-diphosphate is mRNA, including prokaryotic mRNA. Insome embodiments in which the primary RNA is from an RNA amplificationreaction, the RNA amplification reaction is a sense RNA amplificationreaction, such as, but not limited to a sense RNA amplification reactionreferenced herein above. However, in some embodiments, the RNAamplification reaction used to obtain the primary RNA is an anti-senseRNA amplification reaction, such as, but not limited to an anti-senseRNA amplification reaction referenced herein above. In some embodimentsof the kit or method, the affinity tag having the reactive moiety is abiotinylation reagent. In some embodiments of the kit or method whereinthe 2′ or 3′ substituent of the modified cap nucleotide is an aminogroup, the reactive moiety is a biotinylation reagent. The biotinylationreagent can have any amine-reactive chemical moiety known in the art,such as, but not limited to, an acylating moiety, such as anN-hydroxysuccinimidyl (i.e., NHS) ester, or an alkylating moiety. Insome embodiments of the kit or method wherein the 2′ or 3′ substituentof the modified cap nucleotide is an amino group, the affinity tagreagent is biotin-XX-NHS (also called biotin-LC-LC-NHS) or biotin-X-NHS(also called biotin-LC-NHS). In some embodiments of the kit or methodwherein the 2′ or 3′ substituent of the modified cap nucleotide is anazido group, the reactive moiety of the affinity tag is a reactivealkynyl group which is capable of joining the affinity binding moleculeto the azido group via a 1,3-dipolar cycloaddition; in embodiments ofthe method, the affinity tag is joined to the modified cap nucleotide ofthe modified-nucleotide-capped RNA via a 1,2,3-triazole moiety. In someembodiments of the kit or method wherein the affinity tag comprisesbiotin, the affinity-tag-binding molecule is labeled streptavidin oravidin that is labeled with a phycobiliprotein, a dye or any otherdetectable molecule known in the art.

The invention also comprises kits and methods for releasing the labelfrom the labeled modified-nucleotide-capped RNA. For example, someembodiments of the kit additionally comprise a pyrophosphatase, such as,but not limited to tobacco acid pyrophosphatase, or a decapping enzyme,such as, but not limited to yeast or human decapping enzyme. Someembodiments of the method further comprise the step of contacting thelabeled modified-nucleotide-capped RNA that is labeled with thepyrophosphatase, such as tobacco acid pyrophosphatase, or with thedecapping enzyme, such as yeast or human decapping enzyme, whereby thelabeled modified cap nucleotide is cleaved from the RNA and the label isreleased.

The invention also provides kits and methods for direct labeling ofuncapped RNA comprising primary RNA transcripts or RNA having a5′-diphosphate, whether uncapped RNA comprising primary RNA transcriptsor RNA having a 5′-diphosphate is obtained from a biological sample,such as a prokaryotic cell, or a eukaryotic cell or tissue that isinfected by a prokaryote, or are synthesized in vitro, such as in an invitro transcription or RNA amplification reaction, with a detectablemolecule, such as, but not limited to, a visible, fluorescent,luminescent, or infrared fluorescent dye. Some embodiments provide a kitfor direct labeling of uncapped RNA comprising primary RNA transcriptsor RNA having a 5′-diphosphate, the kit comprising: a modified capnucleotide selected from the group consisting of a guaninenucleoside-5′-triphosophate having 2′ or 3′ substituent consisting of anamino or an azido group; and a detectable molecule having a reactivemoiety. Some embodiments provide a method for direct labeling of theuncapped RNA comprising primary RNA transcripts or RNA having a5′-diphosphate, the method comprising: (i) providing an uncapped RNAcomprising primary RNA transcripts or RNA having a 5′-diphosphate, acapping enzyme system, a modified cap nucleotide having 2′ or 3′substituent consisting of an amino or an azido group, and a detectablemolecule having a reactive moiety; (ii) contacting the uncapped RNAcomprising primary RNA transcripts or RNA having a 5′-diphosphate withthe capping enzyme system and the modified cap nucleotide underconditions wherein the uncapped RNA comprising primary RNA transcriptsor RNA having a 5′-diphosphate is capped and amodified-nucleotide-capped RNA is synthesized; and (iii) contacting themodified-nucleotide-capped RNA with the detectable molecule having areactive moiety under conditions wherein the detectable molecule reactswith the modified cap nucleoside of the modified-nucleotide-capped RNAand the RNA is thereby labeled. The primary RNA can be from a biologicalsource or from an in vitro transcription reaction, including an RNAamplification reaction. In preferred embodiments, the RNA amplificationreaction used to obtain the primary RNA is a sense RNA amplificationreaction, such as, but not limited to a sense RNA amplification reactionreferenced herein above. However, in some embodiments, the RNAamplification reaction used to obtain the primary RNA is an anti-senseRNA amplification reaction, such as, but not limited to an anti-senseRNA amplification reaction referenced herein above. In some embodimentsof the kit or method wherein the 2′ or 3′ substituent of the modifiedcap nucleotide is an amino group, the reactive moiety is an acylatinggroup, such as an N-hydroxysuccinimidyl (i.e., NHS) ester, or analkylating group. A wide variety of detectable molecules having areactive group that can react with the 2′ or 3′ amino substituent of themodified cap nucleotide are known in the art, including, but not limitedto visible, fluorescent, luminescent, and infrared fluorescent dyes,such as Cy dyes, fluorescein dyes, rhodamine dyes and the like, and theinvention includes the use of any such detectable molecule in the kitsor methods of the invention. In some embodiments of the kit or methodwherein the 2′ or 3′ substituent of the modified cap nucleotide is anazido group, the reactive moiety of the detectable molecule is areactive alkynyl group which is capable of joining the detectablemolecule to the azido group via a 1,3-dipolar cycloaddition; inembodiments of the method, the detectable molecule is joined to themodified cap nucleotide of the modified-nucleotide-capped RNA via a1,2,3-triazole moiety. Dye molecules having a reactive moiety consistingof an alkynyl group, which can be used for the kits and methods of thepresent invention, are known in the art (e.g., see Breinbauer, R andKohn, M, ChemBioChem 4: 1147-1149, 2003 and references therein), andthose with knowledge in the art will understand how to readily obtain ormake a dye molecule having a reactive alkynyl group using what is known.In preferred embodiments, the reactive detectable dye does not labelgreater than ten percent of RNA molecules that do not have the modifiedcap nucleoside with the respective amino, azido, or thiol substituent.More preferably, the reactive detectable dye labels less than fivepercent or less than one percent of the RNA molecules that do not havethe modified cap nucleoside with the respective amino, azido, or thiolgroup. Most preferably, the reactive detectable dye labels less than onepercent of the RNA molecules that do not have the modified capnucleoside with the respective amino, azido, or thiol group.

The invention also comprises kits and methods for releasing the labelfrom the labeled modified-nucleotide-capped RNA. For example, someembodiments of the kit additionally comprise a pyrophosphatase, such as,but not limited to tobacco acid pyrophosphatase, or a decapping enzyme,such as, but not limited to yeast or human decapping enzyme. Someembodiments of the method further comprise the step of contacting thelabeled modified-nucleotide-capped RNA with a pyrophosphatase, such astobacco acid pyrophosphatase, or a decapping enzyme, such as, but notlimited to yeast or human decapping enzyme, whereby the labeled modifiedcap nucleotide is cleaved from the RNA and the label is removed.

The kits and methods for labeling uncapped RNA comprising primary RNAtranscripts or RNA having a 5′-diphosphate, and in some embodiments, forreleasing the labeled modified cap nucleotide from labeledmodified-nucleotide-capped RNA can be used as assays for detecting thepresence and quantifying the amount of one or more uncapped RNAscomprising primary RNA transcripts or RNA having a 5′-diphosphate in asample. Thus, some embodiments of the method additionally comprises thestep of measuring the amount of label attached to themodified-nucleotide-capped RNA or measuring the amount of label releasedfrom the modified-nucleotide-capped RNA in a sample upon treatment witha pyrophosphatase or a decapping enzyme, thereby detecting and/orquantifying the amount of modified-nucleotide-capped RNA present in thesample. In some embodiments, the labeled modified-nucleotide-capped RNAis used as labeled target for hybridization to microarrays ofoligonucleotides or polynucleotides corresponding to genes and/ortranscripts for a particular cell, tissue or organism (e.g., Affymetrixor Illumina microarrays).

Cap-Labeling: Methods and Kits for Labeling Uncapped RNA

Still other embodiments of the invention provide a cap-labeling methodfor labeling uncapped RNA comprising primary RNA or RNA having a5′-diphosphate in a sample, the method comprising: (i) providing asample comprising the uncapped RNA; a capping enzyme system; a modifiedcap nucleotide, wherein the modified cap nucleotide contains a chemicalbinding moiety to facilitate binding to an affinity-tag-bindingmolecule; and the affinity-tag-binding molecule to which a detectablelabel is attached; (ii) contacting the uncapped RNA with the cappingenzyme system and the modified cap nucleotide under conditions whereinmodified-nucleotide-capped RNA is synthesized; (iii) contacting themodified-nucleotide-capped RNA with reagents and under conditions thatfacilitate binding of the modified cap nucleotide to theaffinity-tag-binding molecule to which the detectable label is attached;and (iv) contacting the modified-nucleotide-capped RNA to theaffinity-tag-binding molecule to which the detectable label is attached,thereby labeling the modified-nucleotide-capped RNA. In some embodimentsof this cap-labeling method, the chemical binding moiety of the modifiedcap nucleotide provided in step (i) comprises an affinity tag that iscapable of binding the affinity-tag-binding molecule to which thedetectable label is attached and step (iii) comprises incubating themodified-nucleotide-capped RNA in a buffer and under conditions thatfacilitate binding of the modified cap nucleotide to theaffinity-tag-binding molecule to which the detectable label is attached.In some other embodiments of the cap-labeling method, the modified capnucleotide provided in step (i) contains a chemical binding moietycomprising an amino, an azido, or a thiol group and step (iii) comprisescontacting the modified-nucleotide-capped RNA with an affinity tagreagent under conditions wherein the affinity tag is chemically joinedto the chemical binding moiety, and further incubating themodified-nucleotide-capped RNA in a buffer and under conditions thatfacilitate binding of the modified cap nucleotide to theaffinity-tag-binding molecule to which the detectable label is attached.Thus, one embodiment of the invention comprises a kit for cap-labeling,the kit comprising: (i) a capping enzyme system; (ii) a modified capnucleotide, wherein the modified cap nucleotide contains a chemicalbinding moiety to facilitate binding to an affinity-tag-bindingmolecule; and (iii) the affinity-tag-binding molecule to which adetectable label is attached. In some embodiments of the kit forcap-labeling, the chemical binding moiety of the modified cap nucleotidecomprises an affinity tag that is capable of binding theaffinity-tag-binding molecule. In other embodiments of the kit forcap-labeling, the chemical binding moiety of the modified cap nucleotidecomprises an amino, an azido, or a thiol group, and the kit additionallycomprises: (iv) an affinity tag reagent that is capable of reacting withthe respective amino, azido, or thiol group of the modified capnucleotide, thereby chemically joining the affinity tag to the chemicalbinding moiety of the modified cap nucleotide. In preferred embodimentsof the methods and kits for cap-labeling, the affinity tag is biotin andthe affinity-tag-binding molecule is streptavidin or avidin.

The biotinylated modified-nucleotide-capped RNA is labeled by binding tostreptavidin or avidin that is covalently attached to a detectablemolecule, including, without limit, a visible, fluorescent, luminescent,or infrared fluorescent molecule, such as, but not limited to,phycoerythrin or another phycobiliprotein, fluorescein, rhodamine, Cy3,Cy5, or an Alexa dye, or another dye, or to streptavidin or avidin thatis covalently attached to an enzyme that is detectable using asubstrate, such as, but not limited to, a substrate that results in avisible, fluorescent, luminescent, or infrared fluorescent signal underenzymatic reaction conditions. The binding of the labeled streptavidinor avidin affinity-tag-binding molecule to the biotinylatedmodified-nucleotide-capped RNA provides a detection method for uses suchas, but not limited to, detecting a nucleic acid sequence as part of adiagnostic assay or for assay of gene expression on an array ormicroarray.

In some embodiments, the method for cap-labeling further comprises thestep of: (v) contacting the modified-nucleotide-capped RNA that is boundto the surface with a protein or biochemical reagent under conditionswherein the triphosphate between the modified cap nucleotide and the5′-penultimate nucleotide of the modified-nucleotide-capped RNA iscleaved, thereby de-capping the modified-nucleotide-capped RNA andreleasing the label therefrom. In some preferred embodiments, theprotein or biochemical reagent that cleaves the triphosphate is apyrophosphatase or decapping enzyme. In some preferred embodiments, theprotein or biochemical reagent that cleaves the triphosphate is selectedfrom the group consisting of tobacco acid pyrophosphatase, Saccharomycescerevisiae decapping enzyme, and human decapping enzyme. Thus, in someembodiments of the kit for cap-labeling, the kit additionally comprises:(v) a protein or biochemical reagent that is capable of specificallycleaving the triphosphate in the modified-nucleotide-capped RNA betweenthe modified cap nucleotide and the 5′-penultimate nucleotide of themodified-nucleotide-capped RNA without cleaving other positions in themodified-nucleotide-capped RNA. In some preferred embodiments of thekit, the protein or biochemical reagent that is capable of specificallycleaving the triphosphate selected from the group consisting of tobaccoacid pyrophosphatase, Saccharomyces cerevisiae decapping enzyme, andhuman decapping enzyme.

In still another embodiment for labeling uncapped RNA, the basic methodof the invention is a direct method for labeling uncapped RNA, themethod comprising: (i) providing a capping enzyme system, and a modifiedcap nucleotide; and (ii) contacting the uncapped RNA with a cappingenzyme system and a modified cap nucleotide under conditions wherein amodified-nucleotide-capped RNA is synthesized. In this embodiment, themodified cap nucleotide itself comprises a guaninenucleoside-5′-triphosphate wherein the detectable label is joined to aposition comprising the 2′- or 3′-amino, azido, or thiol group of thesugar, or to the O6-position of the guanine base, or to the S6-positionof the thioguanine base. A kit of this embodiment comprises: (i) thecapping enzyme system; and (ii) the modified cap nucleotide comprisingthe detectable label.

In specific embodiments of the method in which the modified capnucleotide comprises a 2′- or 3′-amino-, azido-, or mercapto-modifieddeoxyguanosine-5′-triphosphate (e.g., 2′-amino-2′-dGTP,3′-amino-3′-dGTP, 2′-amino-2′,3′-ddGTP, 3′-amino-2′,3′-ddGTP,2′-azido-2′-dGTP, 3′-azido-3′-dGTP, 2′-azido-2′,3′-ddGTP,3′-azido-2′,3′-ddGTP, 2′-mercapto-2′-dGTP, 3′-mercapto-3′-dGTP,2′-mercapto-2′,3′-ddGTP, 3′-mercapto-2′,3′-ddGTP), the method furthercomprises the step of reacting the modified-nucleotide-capped RNA with areactive detectable dye under conditions wherein the amino, azido, ormercapto group is labeled with the dye under conditions whereindetectable modified-nucleotide-capped RNA is obtained. Thus, in someembodiments of the method in which the modified cap nucleotide is anamino-modified deoxyguanosine-5′-triphosphate, the method furthercomprises reacting the modified-nucleotide-capped RNA with a reactivedetectable dye, such as, but not limited to, a fluorescent dye, aluminescent dye, a visible dye, or infrared fluorescent dye, such asselected from the group consisting of a N-hydroxysuccinimidyl ester of aCy dye, a rhodamine dye, a fluorescein dye, and another dye that isknown in the art. In some embodiments wherein the modified capnucleotide is an azido-modified deoxyguanosine-5′-triphosphate, themethod further comprises reacting the modified-nucleotide-capped RNAwith a detectable dye having a reactive alkyne moiety. In someembodiments wherein the modified cap nucleotide is a mercapto-modifieddeoxyguanosine-5′-triphosphate, the method further comprises reactingthe modified-nucleotide-capped RNA with a detectable dye having areactive moiety, such as a maleimidyl moiety. Thus, the method of thepresent invention also provides a way to label uncapped RNA comprisingprimary RNA transcripts or RNA having a 5′-diphosphate with a detectabledye. Any detectable molecule known in the art can be used in the presentinvention. Examples of some detectable molecules which can be used aredescribed in “Handbook of Fluorescent Probes and Research Products”,Ninth Edition, by R. P. Hoagland, Molecular Probes, Inc.

Kits

The present invention also provides kits comprising a capping enzymesystem and a modified cap nucleotide of the present invention.Additionally, the present invention provides kit having one or morecomponents useful, sufficient, and/or necessary for carrying out any ofthe methods described herein. In some embodiments, the kit comprisespoxvirus capping enzyme and a modified cap nucleotide. In someembodiments, the kit comprises vaccinia virus capping enzyme system anda modified cap nucleotide of the present invention. Some embodiments ofthe kit comprise a vaccinia virus capping enzyme system purified fromvaccinia virions, whereas other embodiments of the kit comprise avaccinia virus capping enzyme system from a recombinant source. Thepresent invention includes embodiments of the kit, wherein the modifiedcap nucleotide is a 2′- or 3′-modified nucleotide. In some embodimentsof the kit of the invention, the modified cap nucleotide is a guaninenucleoside-5′-triphosphate wherein the 2′- or 3′-position of the ribosesugar moiety is substituted with a group such as, but not limited to, anO-methyl group, an amino group, an azido group or a fluorine group. Byway of example, but without limitation, in some embodiments of the kit,the modified cap nucleotide is selected from the group consisting of:guanine 3′-O-methyl-ribonucleoside-5′-triphosphate; guanine2′-O-methyl-ribonucleoside-5′-triphosphate; guanine2′-amino-2′-deoxyribonucleoside-5′-triphosphate; guanine3′-amino-3′-deoxyribonucleoside-5′-triphosphate; guanine2′-azido-2′-deoxyribonucleo-side-5′-triphosphate; guanine3′-azido-3′-deoxyribonucleo-side-5′-triphosphate; or guanine2′-fluoro-2′-deoxyribonucleoside-5′-triphosphate. In some embodiments ofthe kit, the modified cap nucleotide is guanine2′-deoxyribonucleoside-5′-triphosphate or guanine3′-deoxyribonucleoside-5′-triphosphate.

In some embodiments of the kit, the modified cap nucleotide is a guaninenucleoside-5′-triphosphate wherein the base is modified. By way ofexample, but without limitation, in some embodiments of the method, themodified cap nucleotide is selected from the group consisting ofN¹-methylguanine-ribonucleoside-5′-triphosphate andO⁶-methylguanine-ribonucleoside-5′-triphosphate. The kit of theinvention is not limited to these modified cap nucleotides. The modifiedcap nucleotide in the kit can be any modified guaninenucleoside-5′-triphosphate that is compatible with the enzymaticactivities of the capping enzyme systems of the present invention.

In some embodiments, the kit further comprises S-adenosyl-methionine orS-adenosyl-ethionine. In some embodiments, the co-factor (e.g.,S-adenosyl-methionine) is used with an enzyme havingguanine-7-methyltransferase activity, such as, but not limited to, theguanine-7-methyltransferase that comprises the vaccinia capping enzymesystem.

In some embodiments, the kit further comprises an mRNA(nucleoside-2′-O—) methyltransferase. In some embodiments, the mRNA(nucleoside-2′-O—) methyltransferase activity is encoded by poxvirusDNA. In some embodiments, the mRNA (nucleoside-2′-O—) methyltransferaseactivity is encoded by vaccinia virus DNA. In such embodiments, the kitcan also further comprise S-adenosyl-methionine.

In some embodiments, the kit further comprises a poly(A) polymerase andother reagents, including ATP, for polyadenylation of RNA.

In some embodiments, the kit further comprises a cell-free extract forin vitro translation. In some embodiments, the cell-free extract isselected from the group consisting of an animal, a plant, a yeast, and ahuman cell-free extract. In some embodiments, the cell-free extract isselected from the group consisting of a rabbit reticulocyte lysate, awheat germ lysate, a drosophila embryo lysate, and a human reticulocytelysate, wherein the human reticulocytes are derived from human stemcells in culture. In some embodiments, the human reticulocytes areprepared from embryonic stem cells. In other embodiments, the humanreticulocytes are prepared from adult stem cells from a patient with acondition. In other embodiments, the human reticulocytes are preparedfrom adult stem cells from a donor.

In some embodiments, the kit further comprises reagents fortransformation or transfection of a eukaryotic cell. Thus, in someembodiments, the kit further comprises reagents for transfectionmethods, such as, but not limited to, cationic lipids for lipid-mediatedtransfection, or solutions for electroporation, or calcium phosphatetransfection. In preferred embodiments, the kit further comprisesreagents for transformation or transfection of a dendritic cell, amacrophage cell, an immune system cell, an epithelial cell, or anartificially generated APC.

In some embodiments wherein the modified cap nucleotide is2′-amino-2′-dGTP or 3′-amino-3′-dGTP, the kit further comprises abiotinylation reagent. By way of example, but without limitation, insome embodiments, the biotinylation reagent is biotin-X-X-NHS orbiotin-X-NHS. In some such embodiments, the kit can further comprisestreptavidin or avidin. In some embodiments, the streptavidin or avidinis attached to a surface. In other such embodiments, the kit can furthercomprise a reactive fluorescent, infrared fluorescent, visible, or otherdetectable dye, such as, but not limited to, an N-hydroxysuccinimidylester of a Cy dye, a fluorescein dye, a rhodamine dye or an Alexa dye.In other embodiments, the reagent compound in the kit can comprise anaffinity tag other than biotin that has a moiety for binding themodified-nucleotide-capped RNA with an affinity-tag-binding molecule ora detectable dye for labeling the modified-nucleotide-capped RNA withthe dye, or it can provide another functionality. In some embodiments,the kit additionally comprises an affinity-tag-binding molecule, whichis either free or attached to a surface.

In some embodiments wherein the modified cap nucleotide in the kit is2′-azido-2′-dGTP or 3′-azido-3′-dGTP, the kit further comprises areagent compound containing an alkynyl or acetylene moiety that canreact with the azido group of the modified capped nucleotide, therebyforming a modified-nucleotide-capped RNA that is derivatized with thereagent compound. The reagent compound can comprise an affinity taghaving a moiety for binding the modified-nucleotide-capped RNA with anaffinity-tag-binding molecule or a detectable dye for labeling themodified-nucleotide-capped RNA with the dye, or it can provide anotherfunctionality. In some embodiments, the kit additionally comprises anaffinity-tag-binding molecule, which is either free or attached to asurface. In other embodiments, the kit can further comprise a reactive(e.g., having an alkynyl group) fluorescent, infrared fluorescent,visible, or other detectable dye, such as, but not limited to, a Cy dye,a fluorescein dye, a rhodamine dye or an Alexa dye having an alkynylgroup.

In some embodiments in which the kit comprises a modified cap nucleotideother than an amino- or azido-modified dGTP, the kit further comprises areagent compound that has a reactive moiety or an affinity bindingmolecule, which moiety or affinity binding molecule can react with orbind with the modified capped nucleotide under conditions wherein aderivatized modified-nucleotide-capped RNA is obtained. The reagentcompound can also provide another moiety for binding and capturing orfor labeling the modified-nucleotide-capped RNA with a detectable dye,or it can provide another functionality.

In some such embodiments, the kit can further comprise streptavidin oravidin, which is either free or attached to a surface. In other suchembodiments, the kit can further comprise a reactive detectable dye,such as, but not limited to, a fluorescent dye selected from the groupconsisting of a N-hydroxysuccinimidyl ester of a Cy dye.

In some embodiments, the kit comprises an RNase. In some embodiments theRNase is RNase I or RNase A. In some embodiments, the kit furthercomprises an RNase H. In some embodiments, the kit comprises a buffer.In some embodiments, the kit comprises reagents for in vitrotranscription of RNAs. In some embodiments, the kit comprises reagentsfor using the capped RNA of the present invention in RNA vaccineapplications (e.g., buffers, cells, transfection reagents, controlreagents, etc.).

In some embodiments, the kit further comprises a cell (e.g., a dendriticor macrophage cell) that is transformed or transfected withmodified-nucleotide-capped RNA having either a cap 0 or a cap Istructure, which modified-nucleotide-capped RNA is preferably alsopolyadenylated. In some embodiments the cell has been transformed ortransfected with modified-nucleotide-capped RNA that is prepared usingRNA from an in vitro transcription reaction or an RNA amplificationreaction, and then capped with a modified cap nucleotide, andpolyadenylated using methods of the present invention.

In some embodiments, the present invention provides a kit for catalyzingformation of a modified-nucleotide-capped RNA comprising: a polypeptidesequence for full-length vaccinia virus capping enzyme, or anenzymatically active portion thereof, and a modified cap nucleotide. Insome embodiments, the kit additionally comprises one or more of aribonuclease inhibitor, an amino acid mixture, ATP, S-adenosylmethioninefor methylation (e.g., of guanine residues), and/or magnesium salt.

In some embodiments, the present invention provides a kit for producingprotein (e.g., from a DNA template via sequential transcription,catalyzed formation of a modified-nucleotide-capped RNA, andtranslation), the kit comprising: (a) a polypeptide sequence forvaccinia virus capping enzyme, and a modified cap nucleotide; (b) acell-free cell extract (e.g., eukaryotic cell-free extract from plant,animal or yeast cells); (c) ribonucleotide triphosphates; and (d) RNApolymerase. In some embodiments, the kit is configured to generate RNAfrom any DNA template. In some embodiments, the DNA template isdownstream of a T7, T3 or SP6 RNA polymerase promoter. In someembodiments, the DNA template includes sequence in a linearized plasmid,cDNA, a double-stranded oligo, or PCR product.

In some embodiments, the present invention provides a kit for producingprotein (e.g., from an uncapped RNA template via coupled catalyzedformation of modified-nucleotide-capped RNA and translation), the kitcomprising: (a) a polypeptide sequence for vaccinia virus cappingenzyme, and a modified cap nucleotide; and (b) cell-free extract (e.g.,eukaryotic cell-free extract from plant, animal or yeast cells). In someembodiments, the cell-free extract is rabbit reticulocyte lysate. Insome embodiments, the cell-free extract is drosophila embryo extract. Insome embodiments, the cell-free extract is wheat germ extract. In someembodiments, the cell-free extract is a human reticulocyte lysate. Insome embodiments, the cell-free extract is synthetic (e.g., noteukaryotic cell-derived).

In some embodiments, all kit components are present within a singlecontainer (e.g., vial or tube). In some embodiments, each kit componentis located in a single container (e.g., vial or tube). In someembodiments, one or more kit components are located in a singlecontainer (e.g., vial or tube) with other components of the same kitbeing located in a separate container (e.g., vial or tube). For example,in some embodiments, a kit comprises a container comprising a solutionof modified cap nucleotide and one or more other containers, wherein oneof the other containers comprises a poxvirus capping enzyme. In someembodiments, a kit comprises instructions for use of the kit. In someembodiments, a kit of the present invention comprises reagents (e.g.,poly(A) polymerase and/or ATP) for polyadenylation of RNA. In someembodiments, a kit of the present invention comprises ribonucleoside5′-triphosphates (e.g., ATP, CTP, GTP, or UTP (e.g., each in their owncontainer or, as a premixed solution of all four).

In some embodiments, a kit of the present invention comprises a solutionof the modified cap nucleotide. In some embodiments, a kit comprises abuffer. In some embodiments, a kit of the present invention comprises oris used with biotin-labeled nucleoside triphosphates and/oraminoallyl-labeled nucleoside triphosphates. In some embodiments, a kitof the present invention comprises a radiolabeled agent (e.g.,³H-methyl-S-adenosyl-L-methionine, ¹⁴C-methyl-S-adenosyl-L-methionine,or α-³²P-GTP). In some embodiments, a kit of the present inventioncomprises an RNase inhibitor. In some embodiments, a kit of the presentinvention may include a DNase (e.g., DNase I, e.g., RNase-free DNase I,including DNase I from bovine pancreas). In some embodiments, a kitcomprises components for an RNA amplification reaction (e.g., foramplification of mRNA from laser capture microdissection or “LCM”samples or from other samples of limited quantity).

The uncapped RNAs comprising primary RNA transcripts or RNAs having a5′-diphosphate that are capped using a method or kit of the inventioncan be of any length. For example, in some embodiments, RNA capped bythe compositions and methods of the present invention is 0-0.1 kB inlength. In some embodiments, the RNA is between 0.1-0.5 kB in length. Insome embodiments, the RNA is between 0.5-1.0 kB in length. In someembodiments, the RNA is between 1-2 kB in length. In some embodiments,the RNA is between 2 kB-10 kB in length. In some embodiments, the RNA isbetween 0.1 kB-10 kB in length. In some embodiments, the RNA is longerthan 10 kB in length.

In some embodiments, the present invention provides nucleic acidsencoding one or more enzymatic activities of a capping enzyme system,together with plasmids, vectors and/or cells for genetic complementationassays in order to identify, screen, correct and/or monitor a geneticdefect in the capping pathway. In some embodiments, the nucleic acidsequence encodes poxvirus capping enzyme and also comprises vectorexpression sequences and/or regulatory element sequences. In someembodiments, the nucleic acid sequence encodes vaccinia virus cappingenzyme and also comprises vector expression sequences and/or regulatoryelement sequences. In some embodiments, the nucleic acid encoding one ormore activities of a capping enzyme system (e.g., a nucleic acidsequence that encodes vaccinia virus capping enzyme) are in anartificial transposon, such as an EZ-Tn5 transposon or a HyperMutransposon. In some embodiments, said artificial transposon comprises asynaptic complex between the transposon and a transposase thatrecognizes the transposon recognition sequences in said transposon(i.e., a “transposome”), such as an EZ-Tn5 or HyperMu transposome.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described compositions and methods of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention that are obvious to thoseskilled in the relevant fields are intended to be within the scope ofthe present invention.

EXAMPLES

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

A. Methods Method 1. Reaction Mixture for Evaluation ofNucleoside-5′-Triphosphates as Substrates for Capping Enzyme: Synthesisof Capped RNA Having a Cap 0 Structure, but Lacking an N⁷-Methyl Groupin the Cap Nucleotide

In order to evaluate the ability of a nucleoside-5′-triphosphate to beused as a modified cap nucleotide substrate by capping enzyme forsynthesis of modified-nucleotide-capped RNA having a cap 0 structurelacking a 7-methylguanine in the cap nucleotide, capping enzymereactions were prepared that contained: 1 μg of a 51-base primary RNAtranscript (prepared by in vitro transcription of a T7promoter-containing double-stranded DNA template using a AmpliScribe™T7-Flash™ Transcription Kit according to the protocol provided with thekit from EPICENTRE Biotechnologies, Madison, Wis., USA) as substrate; 1×Capping Enzyme Buffer (50 mM Tris-HCl, pH 8.0; 6 mM KCl; 1.25 mM MgCl₂);1 mM of each respective nucleoside-5′-triphosphate tested; 0.1 μg ofvaccinia capping enzyme (10 GTPase units); and water to a final reactionvolume of 20 μl. One GTPase unit catalyzes release of one nanomole ofinorganic phosphate from GTP in 10 minutes at 37° C. under standardassay conditions.

Method 2. Reaction Mixture for Evaluation of Nucleoside-5′-Triphosphatesas Substrates for Capping Enzyme Systems for Synthesis of Capped RNA orModified-Nucleotide-Capped RNA Having a Cap 0 Structure with anN⁷-Methyl Group in the Cap Nucleotide

In order to evaluate the ability of a nucleoside-5′-triphosphate to beused as a modified cap nucleotide substrate by a capping enzyme systemfor synthesis of modified-nucleotide-capped RNA having a cap 0 structurewith a 7-methyl group in the base of the cap nucleotide, capping enzymereactions were prepared as described in Method 1 above, except that thereaction mixture additionally contained: 0.25 μCi(¹⁴C-methyl)-S-adenosyl-L-methionine (specific activity: 55 Ci/mmol).

Method 3. Capping Enzyme System Reactions.

Unless otherwise stated, each capping enzyme reaction prepared asdescribed in Method 1 was incubated at 37° C. for 30 minutes. Unlessotherwise stated, each capping enzyme reaction prepared as described inMethod 2 was incubated at 37° C. for 2 hours. All reactions were storedat −20° C. until analyzed.

Method 4. PAGE Analysis of Results of Capping Enzyme System and OtherReactions.

An aliquot corresponding to 0.1 μg of RNA (i.e., 2 μl) from eachcompleted capping enzyme reaction was subjected to denaturingpolyacrylamide gel electrophoresis on a 20% polyacrylamide, 8 M Urea,1×TBE gel. The gel was run at 510 volts for 4 hours, then stained byethidium bromide and photographed.

Method 5. Autoradiography of PAGE Gels.

When assays using radioactive S-adenosyl-L-methionine were performedaccording to Method 2 or Method 7, the PAGE gel was subsequently drieddown and exposed to film for 7-10 days in order to visualizeradioactively-labeled reaction products by autoradiographic detection.

Method 6. Preparation of Modified-Nucleotide-Capped RNA Having aModified Cap Nucleotide with an N⁷-Methyl Group as Substrates forSynthesis by mRNA (Nucleoside-2′-O—) Methyltransferase ofModified-Nucleotide-Capped RNA Having a Cap I Structure.

In order to assay for the ability of a modified-nucleotide-capped RNA(or a standard unmodified m⁷G-capped RNA control) having a cap 0structure, wherein the cap nucleotide has a methyl group on theN⁷-position of the nucleic acid base, to be converted to amodified-nucleotide-capped RNA having a cap I structure (i.e., having a2′-O-methyl group on the penultimate nucleotide at the 5-end), 6 μg of a51-base primary RNA transcript was capped in the presence of either GTPor a modified nucleoside-5′-triphosphate selected from the groupconsisting of N¹-methyl-GTP, O⁶-methyl-GTP, 3′-OMe-GTP, 2′-OMe-GTP,2′-dGTP, 2′-F-dGTP, 2′-amino-dGTP, and 2′-azido-dGTP in a capping enzymereaction mixture as described in Method 1, except that the cappingenzyme reaction additionally contained 1 mM unlabeledS-adenosyl-L-methionine. The reaction mixture was incubated at 37° C.for two hours. The reaction products were analyzed as described inMethod 4. The 51-base primary RNA transcript was converted by thecapping enzyme system into a 52-base capped RNA, as determined by PAGEanalysis of the reaction products using 51-base and 52-base size markerelectrophoresis standards. The reaction containing capped RNA was storedat −20° C. until used.

Method 7. Reaction Mixture for Evaluation of Modified-Nucleotide-CappedRNAs Having Different Modified Cap Nucleotides as Substrates for mRNA(Nucleoside-2′-O—) Methyltransferase

In order to evaluate the ability of a modified-nucleotide-capped RNAhaving a cap 0 structure to serve as a substrate for 2′-O-methylation ofthe penultimate nucleotide at the 5-end, two μg of each 52-basemodified-nucleotide-capped RNA (or the unmodified m⁷G-capped RNAcontrol), prepared as described in Method 6, was incubated in a reactionmixture consisting of 1× Reaction Buffer (50 mM Tris-HCl, pH 8.0; 6 mMKCl; 1.25 mM MgCl₂) with 0.220-0.275 μg of vaccinia mRNA(nucleoside-2′-O—) methyltransferase which additionally contained 0.25μCi (¹⁴C-methyl)-S-adenosyl-L-methionine (specific activity: 55Ci/mmol).

Method 8. Assay for Removal of a Modified Cap Nucleotide from aModified-Nucleotide-Capped RNA by Tobacco Acid Pyrophosphatase.

Two μg of a 52-base unmodified m⁷G-capped RNA or amodified-nucleotide-capped RNA obtained in Method 6 was treated withtobacco acid pyrophosphatase (TAP) in 1× reaction buffer as described bythe supplier (EPICENTRE Biotechnologies, Madison, Wis., USA). TheTAP-treated reaction product was analyzed by denaturing polyacrylamidegel electrophoresis on a 20% polyacrylamide, 8 M Urea, 1×TBE gel. Thegel was run at 510 volts for 4 hours, then stained by ethidium bromideand photographed.

Method 9. Polyadenylation of Modified-Nucleotide-Capped RNA UsingPoly(A) Polymerase.

One μg of a 52-base unmodified m⁷G-capped RNA or amodified-nucleotide-capped RNA obtained as described in Method 6 isincubated in the presence of 4 units of A-Plus™ Poly(A)-Polymerase and 1mM ATP in 1× reaction buffer (50 mM Tris-HCL, pH 8.0, 250 mM NaCl, and10 mM MgCl₂, as described by the supplier (EPICENTRE Biotechnologies,Madison, Wis., USA). The polyadenylated reaction product is analyzed bydenaturing polyacrylamide gel electrophoresis on a 20% polyacrylamide, 8M Urea, 1×TBE gel. The gel is run at 510 volts for 4 hours, and then isstained by ethidium bromide and photographed.

Method 10. Synthesis of Modified-Nucleotide-Capped Polyadenylated RNAswith a Cap 0 or a Cap I Structure and Control Capped RNAs from a PrimaryTranscript Obtained by In Vitro Transcription of a DNA TemplateComprising a Gene Located Downstream of a T7 RNA Polymerase Promoter.

pRL-SV40 DNA (Promega, Madison, Wis., USA) was digested to completionwith Xba I endonuclease (New England BioLabs, Ipswich, Mass., USA) asper manufacturer's instructions. The resultant linear double-strandedDNA contains the coding region (gene) for Renilla luciferase downstreamof a phage T7 RNA polymerase promoter. The DNA was used as a templatefor in vitro transcription using an AmpliScribe™ T7-Flash™ TranscriptionKit (EPICENTRE Biotechnologies, Madison, Wis., USA) as permanufacturer's instructions to produce an ˜953-base run-off RNAtranscript. One hundred and twenty micrograms (120 μg) of purifiedRenilla luciferase RNA was poly(A)-tailed using an A-Plus™Poly(A)-Tailing Kit (EPICENTRE Biotechnologies, Madison, Wis., USA) asper manufacturer's instructions except the total reaction volume was 200μl and the incubation was carried out for 60 minutes. Primary RNAtranscripts having poly(A)-tails that were estimated to be 150-200 A'slong were obtained based on gel analysis using size standards. A 7.5-μgsample of the purified T7-transcribed poly(A)-tailed RNA primarytranscript of the Renilla luciferase gene was then capped in a reactionmixture containing 1 mM of one of each of thenucleoside-5′-triphosphates tested in 1× capping buffer (50 mM Tris-HCl,pH 8.0; 6 mM KCl; 1.25 mM MgCl₂) with 1 mM S-adenosyl-methionine (SAM)and 0.2 μg of vaccinia capping enzyme (20 GTPase units) (EPICENTREBiotechnologies, Madison, Wis., USA) for 3 hours at 37° C. Thistreatment produces capped poly(A)-tailed RNA having a cap 0 structure onthe 5′-end. Similarly, in other reactions, a 7.5-μg sample of thepurified T7-transcribed poly(A)-tailed RNA primary transcript of theRenilla luciferase gene was capped in a reaction mixture containing 1 mMof one of each respective nucleoside-5′-triphosphate tested in 1×capping buffer (50 mM Tris-HCl, pH 8.0; 6 mM KCl; 1.25 mM MgCl₂) and 1mM S-adenosyl-methionine (SAM) and 0.2 μg of vaccinia mRNA(nucleoside-2′-O—)methyltransferase (EPICENTRE Biotechnologies, Madison,Wis., USA) for 3 hours at 37° C. This treatment produces cappedpoly(A)-tailed RNA having a cap I structure on the 5′-end of the RNA.The RNA was purified by phenol-chloroform extraction, ammonium acetateprecipitation, 70% ethanol wash, and resuspended in RNase-free water toa final concentration of 0.2-0.5 μg/μl.

Method 11. Assays of In Vivo Translation of Modified-Nucleotide-CappedRNA in Cells

HeLa cells (ATCC, Manassas, Va., USA), a cervical carcinoma cell line,and BDCM cells (ATCC, Manassas, Va., USA), a lymphoblast cell line withcharacteristics of dendritic cells, were grown under standard conditionsin complete growth medium containing DMEM with 4.5 g/L of glucose, 584mg/L of L-glutamine, and 110 mg/L of sodium pyruvate (Mediatech Inc.,Herndon, Va., USA), 10% fetal bovine serum, 100 U/ml penicillin and 100μg/ml streptomycin. Cells were dispensed into 12-well plates and grownfor 24 hours before transfection. mRNA transfections were performed bylipofection. Transfection reaction mixes were prepared in reduced serummedia Opti-Mem® I (Invitrogen Corp, Carlsbad, Calif., USA). Thetransfection mixes for HeLa cells contained 0.25 μg of each invitro-transcribed Renilla luciferase mRNA, 0.5 μl mRNA Boost Reagent and1 μl TransIT®-mRNA Reagent (Minis Bio Corp, Madison, Wis., USA) inserum-containing media and was incubated with the cells forapproximately 18 hours. The transfection mixes for BDCM cells contained0.5 μg of each in vitro-transcribed Renilla luciferase mRNA, 2.5 μl mRNABoost Reagent and 0.5 μl TransIT®-mRNA Reagent (Minis Bio Corp, Madison,Wis., USA) in serum-containing media and was incubated with the cellsfor 8 hours. Cells were harvested by scraping and were lysed in PassiveLysis Buffer (Promega, Fitchburg, Wis., USA). Aliquots of lysed cellextracts were assayed for luminescence with the Luciferase Assay System(Promega, Fitchburg, Wis., USA) as per manufacturer's instructions on aLuminoskan Ascent Luminometer (Thermo Electron Corp., Waltham, Mass.,USA). Relative light units (RLU) per transfection were normalized by theamount of protein in each sample. Total protein content was assayed foreach extract with Coomassie Plus Bradford assay reagent (Pierce,Rockford, Ill., USA) as per manufacturer's instructions.

Method 12. Assays of In Vitro Translation of Modified-Nucleotide-CappedRNA in Cell-Free Extracts

For in vitro translation assays in rabbit reticulocyte lysates, 0.5 μgof each Renilla luciferase mRNA was denatured at 65° C. for 5 minutesand subsequently translated with the Flexi® Rabbit Reticulocyte LysateSystem (Promega, Fitchburg, Wis., USA) as per manufacturer'sinstructions for 90 minutes at 30° C. For in vitro translation assays inwheat germ extracts (Promega, Fitchburg, Wis., USA), the potassiumconcentration was optimized to 50 mM, and 0.5 μg of each Renillaluciferase mRNA was denatured at 65° C. for 5 minutes and subsequentlytranslated in the wheat germ extract for 90 minutes at 25° C. as permanufacturer's instructions. Aliquots of translation extracts wereassayed for luminescence with the Luciferase Assay System (Promega,Fitchburg, Wis., USA) as per manufacturer's instructions on a LuminoskanAscent Luminometer (Thermo Electron Corp., Waltham, Mass., USA).

Results

Experiment 1. Control Reactions Using GTP as the CappingNucleotide±S-Adenosyl-Methionine for Capping Enzyme Reactions, andSubsequent 2′-O-Methylation and/or Polyadenylation of UnmodifiedG-Capped RNA or Unmodified N⁷-Methyl-G-Capped RNA.

1A. When GTP was used as the nucleoside-5′-triphosphate in a cappingenzyme reaction mixture set up as described in Method 1, and the cappingenzyme reaction was carried out as described in Method 3 and analyzed asdescribed in Method 4, the 51-base primary RNA transcript wasquantitatively converted by the capping enzyme system into a 52-basecapped RNA, as determined by polyacrylamide gel electrophoretic analysisof the reaction products using 51-base and 52-base size markerelectrophoresis standards. This was a control reaction and demonstratedthat the capping enzyme system efficiently catalyzed capping of theprimary RNA transcript into capped RNA having a cap 0 structure, whereinthe cap nucleotide lacked a methyl group on the N⁷ position of theguanine base.

1B. When GTP was used as the nucleoside-5′-triphosphate in a cappingenzyme reaction mixture that additionally contained(¹⁴C-methyl)-S-adenosyl-L-methionine, set up as described in Method 2,and the capping enzyme reaction was carried out as described in Method 3and analyzed as described in Method 4, the 51-base primary RNAtranscript was quantitatively converted by the capping enzyme systeminto a 52-base capped RNA, as determined by polyacrylamide gelelectrophoretic analysis of the reaction products using 51-base and52-base size marker electrophoresis standards, and the 52-base cappedRNA contained the radioactive methyl group based on autoradiography ofthe dried gel as described in Method 5. Thus, this control reactiondemonstrated that the capping enzyme system catalyzed capping of theprimary RNA transcript into capped RNA having a cap 0 structure, whereinthe cap nucleotide was additionally methylated at the N⁷ position of theguanine base by the guanine 7-methyltransferase activity of the cappingenzyme. This reaction served as a control for estimating relativemethylation of the N⁷ position of the cap nucleotide ofmodified-nucleotide-capped RNAs.

1C. When the unlabeled unmodified m⁷G-capped RNA control was incubatedwith mRNA (nucleoside-2′-O—) methyltransferase as described in Method 7,and the product was analyzed by PAGE and autoradiography, as describedin Method 4 and Method 5, a 52-base band that contained radioactivitywas observed. This control reaction demonstrated that mRNA(nucleoside-2′-O—) methyltransferase catalyzed methylation of the2′-hydroxyl of the penultimate nucleotide at the 5-end of unmodifiedm⁷G-capped RNA. This reaction served as a control for estimatingrelative methylation of the 5′-penultimate nucleotide inmodified-nucleotide-capped RNA. In separate reactions, it was alsodetermined that, if the RNA was capped by capping enzyme using GTP inthe absence of S-adenosyl-L-methionine, G-capped RNA was still asubstrate for mRNA (nucleoside-2′-O—) methyltransferase-catalyzedmethylation of the 2′-hydroxyl of the 5′-penultimate nucleotide, basedon incorporation of the radioactive methyl group.

1D. When the unmodified m⁷G-capped RNA was treated with tobacco acidpyrophosphatase (TAP), as described in Method 8, a 51-base band wasobserved on PAGE analysis, as described in Method 4, demonstratingremoval of the m⁷G cap nucleotide.

1E. When the unmodified m⁷G-capped RNA was incubated with poly(A)polymerase, as described in Method 9, a poly(A) tail was added to theRNA, the length of which varied with reaction time, substrateconcentration and enzyme concentration.

Experiment 2. Use of Nucleoside-5′-Triphosphates with Base Modificationsas Substrates for Capping Enzyme, and Uses of Modified-Nucleotide-CappedRNA Obtained Therefrom.

2A. ATP, N⁷-methyl-GTP (m⁷GTP), 2′,3′-ddGTP, 7-deaza-GTP, N¹-methyl-GTP,3′-amino-2′,3′-ddGTP, 3′-azido-2′,3′-ddGTP, and O⁶-methyl-GTP were eachused as the nucleoside-5′-triphosphate in a capping enzyme reactionmixture set up as described in Method 1, and the capping enzyme reactionwas carried out as described in Method 3 and analyzed as described inMethod 4. ATP, N⁷-methyl-GTP, 2′,3′-ddGTP, and 7-deaza-GTP were notsubstrates for the capping enzyme system, since the 51-base primary RNAtranscript was not converted by the capping enzyme system into a 52-basecapped RNA. However, N¹-methyl-GTP, 3′-amino-2′,3′-ddGTP,3′-azido-2′,3′-ddGTP, and O⁶-methyl-GTP were substrates for the cappingenzyme system. The capping enzyme system quantitatively converted the51-base primary RNA transcript to a 52-base capped RNA in a 30-minutereaction in the presence of N¹-methyl-GTP based on PAGE analysis. Underthe same conditions, approximately 70% of the 51-base primary RNAtranscript was converted by the capping enzyme system into a 52-basecapped RNA in the presence of O⁶-methyl-GTP. Thus, capping enzyme canuse nucleoside triphosphates with some, but not all, guanine basemodifications as substrates for synthesis of capped RNA having a cap 0structure, wherein the cap nucleotide lacks a methyl group on the N⁷position of the guanine base.

2B. When O⁶-methyl-GTP was used as the nucleoside-5′-triphosphate in acapping enzyme reaction mixture that additionally contained(¹⁴C-methyl)-S-adenosyl-L-methionine, set up as described in Method 2,and the capping enzyme reaction was carried out as described in Method 3and analyzed as described in Method 4 and Method 5, the 51-base primaryRNA transcript was quantitatively converted by the capping enzyme systeminto a radioactive 52-base capped RNA. Thus, the capping enzyme systemcatalyzed formation of N⁷-methylated capped RNA having a cap 0 structurewhen O⁶-methyl-GTP was used as the modified cap nucleotide. However,under the same reaction conditions with the N¹-methyl-GTP, a 52-baseband was formed, but it did not contain any radioactivity, showing thatN¹-methyl-G-capped RNA was not methylated. Therefore, the N¹-methyl-Gmodified cap nucleotide was not a substrate for subsequent methylationat the N⁷ position by the capping enzyme system.

2C. When either unlabeled N¹-methyl-G-capped RNA or O⁶-methyl-G-cappedRNA was incubated with mRNA (nucleoside-2′-O—) methyltransferase asdescribed in Method 7, and the product was analyzed by PAGE andautoradiography, as described in Method 4 and Method 5, a 52-baseradioactive band was observed. Thus, mRNA (nucleoside-2′-O—)methyltransferase catalyzed methylation of the 2′-hydroxyl of the5′-penultimate nucleotide of both N¹-methyl-G-capped RNA andO⁶-methyl-G-capped RNA (although, as described in 2B above, theO⁶-methyl-G-capped RNA had an N⁷-methyl group on the modified capnucleotide, whereas the N¹-methyl-G-capped RNA did not). Thus, RNAcapped with either N¹-methyl-G or O⁶-methyl-G modified cap nucleotideswere substrates for methylation of the 2′-hydroxyl of the 5′-penultimatenucleotide by mRNA (nucleoside-2′-O—) methyltransferase. However, theamount of radioactivity incorporated into the 52-base band using theO⁶-methyl-G-capped RNA as the substrate indicated that the reaction withthis substrate was not highly efficient.

2D. When the N¹-methyl-G-capped RNA or O⁶-methyl-G-capped RNA wastreated with tobacco acid pyrophosphatase (TAP), as described in Method8, a 51-base band was observed on PAGE analysis, as described in Method4, demonstrating removal of the N¹-methyl-G or O⁶-methyl-G capnucleotide.

Experiment 3. Use of Nucleoside-5′-Triphosphates Having 2′ and/or 3′Modifications of the Sugar Moiety as Substrates for Capping Enzyme, andUses of Modified-Nucleotide-Capped RNA Obtained Therefrom.

3A. The following nucleoside-5′-triphosphates having 2′ and/or 3′modifications of the sugar moiety were each used as the modified capnucleotide in a capping enzyme reaction mixture set up as described inMethod 1, and the capping enzyme reaction was carried out as describedin Method 3 and analyzed as described in Method 4: 2′,3′-dideoxy-GTP(i.e., 2′,3′-ddGTP); 2′-dGTP; 2′-OMe-GTP; 3′-OMe-GTP; 2′-F-dGTP;2′-amino-2′-dGTP (i.e., 2′-amino-dGTP); and 2′-azido-2′-dGTP (i.e.,2′-azido-dGTP). The 2′,3′-ddGTP was not a substrate for the cappingenzyme system, since the 51-base primary RNA transcript was notconverted by the capping enzyme system into a 52-base capped RNA.However, all of the remaining nucleoside-5′-triphosphates having 2′and/or 3′ modifications of the sugar moiety were substrates for thecapping enzyme system. The capping enzyme system quantitativelyconverted the 51-base primary RNA transcript to a 52-base capped RNA ina 30-minute reaction in the presence of 2′-dGTP, 3′-OMe-GTP, and2′-amino-dGTP based on PAGE analysis. Under the same conditions,approximately 50-75% of the 51-base primary RNA transcript was convertedby the capping enzyme system into a 52-base capped RNA in the presenceof 2′-OMe-GTP, 2′-F-dGTP, and 2′-azido-dGTP; when the reaction timeusing these modified cap nucleotides was extended from 30 minutes to 2hours or 6 hours, the percentage of the RNA capped increased stillfurther, being approximately 80-100% in some experiments. Thus, cappingenzyme used all of these modified nucleoside triphosphates, except the2′,3′-ddGTP as substrates for synthesis of capped RNA having a cap 0structure, wherein the cap nucleotide lacked a methyl group on the N⁷position of the guanine base.

3B. When 3′-OMe-GTP, 2′-OMe-GTP, 2′-dGTP, 2′-F-dGTP, 2′-amino-dGTP, or2′-azido-dGTP was used as the nucleoside-5′-triphosphate in a cappingenzyme reaction mixture that additionally contained(¹⁴C-methyl)-S-adenosyl-L-methionine, set up as described in Method 2,and the capping enzyme reaction was carried out as described in Method 3and analyzed as described in Method 4 and Method 5, 95-100% of the51-base primary RNA transcript was converted by the capping enzymesystem into a radioactive 52-base capped RNA in a 2-hour reaction. Thus,the capping enzyme system catalyzed formation of N⁷-methylated cappedRNA having a cap 0 structure when these modified cap nucleotides wereused.

3C. When unlabeled modified-nucleotide-capped RNA was prepared using3′-OMe-GTP, 2′-OMe-GTP, 2′-dGTP, 2′-F-dGTP, 2′-amino-dGTP, or2′-azido-dGTP as the cap nucleotide and then was incubated with mRNA(nucleoside-2′-O—) methyltransferase as described in Method 7, and theproduct was analyzed by PAGE and autoradiography, as described in Method4 and Method 5, a 52-base radioactive band was observed for all of themodified-nucleotide-capped RNAs tested. Thus, modified-nucleotide-cappedRNA obtained using 3′-OMe-GTP, 2′-OMe-GTP, 2′-dGTP, 2′-F-dGTP,2′-amino-dGTP, or 2′-azido-dGTP as the modified cap nucleotides weresubstrates for methylation of the 2′-hydroxyl of the 5′-penultimatenucleotide by mRNA (nucleoside-2′-O—) methyltransferase. Thesemodified-nucleotide-capped RNAs also had a methyl group on theN⁷-position of the modified cap nucleotide from the capping enzymereaction of Method 6. However, the mRNA (nucleoside-2′-O—)methyltransferase can also catalyze methylation of the 2′-hydroxyl ofthe 5′-penultimate nucleotide if the N⁷-position of the modified capnucleotide is not methylated. For example, whenmodified-nucleotide-capped RNA was prepared as described in Method 1using 2′-F-dGTP (except that the reaction was carried out for twohours), then incubated with mRNA (nucleoside-2′-O—) methyltransferase asdescribed in Method 7 and the product was analyzed by PAGE andautoradiography as described in Methods 4 and 5, a 52-base radioactiveband was observed.

3D. Modified-nucleotide-capped RNA was prepared as described in Method 6using 3′-OMe-GTP, 2′-OMe-GTP, 2′-dGTP, or 2′-amino-dGTP as the modifiedcap nucleotide. Modified-nucleotide-capped RNA was prepared as describedin Method 6, except in the absence of S-adenosyl-L-methionine, using2′-F-dGTP as the modified cap nucleotide. When each of thesemodified-nucleotide-capped RNAs was treated with tobacco acidpyrophosphatase (TAP), as described in Method 8, a 51-base band wasobserved on PAGE analysis, as described in Method 4, demonstratingremoval of the respective modified cap nucleotide. The PAGE gel obtainedfor the TAP decapping reaction of the modified-nucleotide-capped RNAprepared as described in Method 6 using 2′-azido-dGTP was notinterpretable, so no conclusion could be made about whether thismodified cap nucleotide could be removed by TAP.

Experiment 4. In Vivo Translation of Modified-Nucleotide-CappedPoly(A)-Tailed RNA Having a Cap 0 or Cap I Structure in HeLa and BDCMCells.

Unmodified- or modified-nucleotide-capped poly(A)-tailed Renillaluciferase RNAs having either a cap 0 or a cap I structure were preparedas described in Method 10 using either GTP or one of the followingmodified nucleoside-5′-triphosphates: N¹-methyl-GTP, O⁶-methyl-GTP,2′-dGTP; 3′-dGTP; 2′-OMe-GTP; 3′-OMe-GTP; 2′-F-2′-dGTP (i.e.,2′-F-dGTP); 2′-azido-2′-dGTP (i.e., 2′-azido-dGTP); and 2′-amino-2′-dGTP(i.e., 2′-amino-dGTP). Each of the modified-nucleotide-cappedpoly(A)-tailed Renilla luciferase RNAs was then evaluated for in vivotranslation in HeLa and BDCM Cells as described in Method 11. For eachcell line, the level of translation obtained using eachmodified-nucleotide-capped poly(A)-tailed Renilla luciferase RNA wascompared to the level of translation obtained using the unmodifiedcapped poly(A)-tailed Renilla luciferase RNA obtained using GTP as thecapping nucleotide. Thus, for each cell line, the relative level oftranslation obtained with the unmodified capped poly(A)-tailed Renillaluciferase RNA was assigned a value of 100% and the level of translationobtained with each modified-nucleotide-capped poly(A)-tailed Renillaluciferase RNA was assigned a relative percent translation valuecompared to that obtained using the unmodified capped poly(A)-tailedRenilla luciferase RNA. The relative percent translation value enablescomparisons of the effects of different cap nucleotides on translationefficiency. However, it should be noted that the amount of in vivotranslation product from capped poly(A)-tailed Renilla luciferase RNA,as measured by the number of relative light units (RLUs) per microgramof total protein per sample, was higher for capped RNAs having a cap Istructure than for capped RNAs having the same cap nucleotide but with acap 0 structure. For example, the RLUs measured in HeLa cells were about2.2 times higher using GTP-capped poly(A)-tailed Renilla luciferase RNAhaving a cap I structure than for GTP-capped poly(A)-tailed Renillaluciferase RNA having a cap 0 structure, even though each reading wasdefined as 100% translation efficiency for the purpose of comparing theeffects of different cap nucleotides on RNA having caps of the same capstructure on translation efficiency. However, the differences in theRLUs for GTP-capped poly(A)-tailed Renilla luciferase RNA having a cap Icompared to a cap 0 structure was less in BDCM cells. Thus, the RLUsmeasured in BDCM cells were only about 1.3 times higher using GTP-cappedpoly(A)-tailed Renilla luciferase RNA having a cap I structure thanusing GTP-capped poly(A)-tailed Renilla luciferase RNA having a cap 0structure. The presence of a cap nucleotide was important for in vivotranslation. For example, no translation was detected using this assaywhen HeLa cells were transfected with uncapped poly(A)-tailed Renillaluciferase RNA.

4A. In Vivo Translation in HeLa Cells.

By comparison with the GTP-capped poly(A)-tailed Renilla luciferase RNAhaving a cap 0 structure (100% translation efficiency), the averagerelative translation efficiency in HeLa cells following transfection bya modified-nucleotide-capped poly(A)-tailed Renilla luciferase RNAhaving a cap 0 was as follows if the RNA was capped using:N¹-methyl-GTP, 3%; O⁶-methyl-GTP, 114%; 2′-dGTP, 64%; 3′-dGTP, 45%;2′-OMe-GTP, 86%; 3′-OMe-GTP, 47%; 2′-F-2′-dGTP (i.e., 2′-F-dGTP), 46%;2′-azido-2′-dGTP (i.e., 2′-azido-dGTP), 66%; and 2′-amino-2′-dGTP (i.e.,2′-amino-dGTP), 172%.

The average relative translation efficiency ofmodified-nucleotide-capped poly(A)-tailed Renilla luciferase RNA havinga cap I structure in HeLa cells was as follows if the RNA was cappedusing: N¹-methyl-GTP, <1%; O⁶-methyl-GTP, 134%; 2′-dGTP, 68%; 3′-dGTP,48%; 2′-OMe-GTP, 104%; 3′-OMe-GTP, 31%; 2′-F-2′-dGTP (i.e., 2′-F-dGTP),54%; 2′-azido-2′-dGTP (i.e., 2′-azido-dGTP), 52%; and 2′-amino-2′-dGTP(i.e., 2′-amino-dGTP), 254%.

In subsequent experiments in which a different luciferase transcript wasprepared from a firefly luciferase gene, and the transcript wassubsequently capped and polyadenylated using the same methods as for theabove modified-nucleotide-capped poly(A)-tailed Renilla luciferase RNA,the average relative translation efficiency of thismodified-nucleotide-capped poly(A)-tailed luciferase RNA having a cap Istructure in HeLa cells was as follows if the RNA was capped using:N¹-methyl-GTP, <1%; O⁶-methyl-GTP, 146%; 2′-dGTP, 61%; 3′-dGTP, 61%;2′-OMe-GTP, 90%; 3′-OMe-GTP, 65%; 2′-F-2′-dGTP (i.e., 2′-F-dGTP), 105%;2′-azido-2′-dGTP (i.e., 2′-azido-dGTP), 64%; and 2′-amino-2′-dGTP (i.e.,2′-amino-dGTP), 43%.

In another set of experiments to determine the effect of the poly(A)tail length on in vivo expression, the present inventors found that theamount of in vivo translation in HeLa cells of one cap 0-typepolyadenylated m⁷G-capped Renilla luciferase RNA or m₂^(7, 3′-O)G-capped Renilla luciferase RNA increased as the poly(A) taillength increased up to about 400 nucleotides. However, with the fireflyluciferase RNA, the amount of in vivo translation in HeLa cells of thecap 0-type polyadenylated RNA did not increase if the length of thepoly(A) tail was greater than about 100 to about 200 nucleotides.

4B. In Vivo Translation in BDCM Cells.

By comparison with the GTP-capped poly(A)-tailed Renilla luciferase RNAhaving a cap 0 structure (100% translation efficiency), the relativetranslation efficiency of modified-nucleotide-capped poly(A)-tailedRenilla luciferase RNA having a cap 0 structure in BDCM cells was asfollows if the RNA was capped using: O⁶-methyl-GTP, 115%; and2′-amino-2′-dGTP (i.e., 2′-amino-dGTP), 150%. Whenmodified-nucleotide-capped Renilla luciferase RNA having a cap 0structure was prepared co-transcriptionally using an m₂ ^(7, 3′-O)GpppGcap analog (ARCA) in a T7 RNA polymerase in vitro transcriptionreaction, and then tailed using poly(A) polymerase as described inMethod 10, the relative translation efficiency of the ARCA-capped RNA inBDCM cells was 130% compared to unmodified capped poly(A)-tailed Renillaluciferase RNA obtained using vaccinia capping enzyme and GTP as thecapping nucleotide.

The relative translation efficiency of modified-nucleotide-cappedpoly(A)-tailed Renilla luciferase RNA having a cap I structure in BDCMcells was as follows if the RNA was capped using: O⁶-methyl-GTP, 117%;and 2′-amino-2′-dGTP (i.e., 2′-amino-dGTP), 177%. Whenmodified-nucleotide-capped Renilla luciferase RNA having a cap Istructure was prepared co-transcriptionally using an m₂ ^(7, 3′-O)GpppGcap analog (ARCA) in a T7 RNA polymerase in vitro transcriptionreaction, and then tailed using poly(A) polymerase as described inMethod 10, the relative translation efficiency of the ARCA-capped RNA inBDCM cells was 102% compared to unmodified capped poly(A)-tailed Renillaluciferase RNA obtained using vaccinia capping enzyme and GTP as thecapping nucleotide.

Experiment 5. In Vitro Translation of Modified-Nucleotide-CappedPoly(A)-Tailed RNA Having a Cap 0 or Cap I Structure in Cell-FreeExtracts.

Unmodified- or modified-nucleotide-capped poly(A)-tailed Renillaluciferase RNAs having either a cap 0 or a cap I structure were preparedas described in Method 10 using either GTP or one of the followingmodified nucleoside-5′-triphosphates: N¹-methyl-GTP, O⁶-methyl-GTP,2′-dGTP; 3′-dGTP; 2′-OMe-GTP; 3′-OMe-GTP; 2′-F-2′-dGTP (i.e.,2′-F-dGTP); 2′-azido-2′-dGTP (i.e., 2′-azido-dGTP); and 2′-amino-2′-dGTP(i.e., 2′-amino-dGTP). Each of the modified-nucleotide-cappedpoly(A)-tailed Renilla luciferase RNAs was then evaluated for in vitrotranslation in a rabbit reticulocye lysate and a wheat germ lysate asdescribed in Method 12. For each lysate, the level of translationobtained using each modified-nucleotide-capped poly(A)-tailed Renillaluciferase RNA was compared to the level of translation obtained usingthe unmodified capped poly(A)-tailed Renilla luciferase RNA obtainedusing GTP as the capping nucleotide. The level of translation obtainedwith the unmodified capped poly(A)-tailed Renilla luciferase RNA in eachlysate was assigned a value of 100% and the level of translationobtained with each modified-nucleotide-capped poly(A)-tailed Renillaluciferase RNA was assigned a relative percent translation value forthat lysate compared to that obtained using the unmodified cappedpoly(A)-tailed Renilla luciferase RNA. The presence of a cap nucleotidewas helpful but not essential for in vitro translation, since the levelof translation of uncapped poly(A)-tailed Renilla luciferase RNA inrabbit reticulocyte lysate and wheat germ lysate was about 50% and 32%,respectively, of that obtained using either a cap 0- or a cap I-typecapped poly(A)-tailed Renilla luciferase RNA made using vaccinia cappingenzyme and GTP as the capping nucleotide.

5A. In Vitro Translation in Rabbit Reticulocyte Lysate.

By comparison with the GTP-capped poly(A)-tailed Renilla luciferase RNAhaving a cap 0 structure (100% translation efficiency), the averagerelative translation efficiency in rabbit reticulocyte lysate ofmodified-nucleotide-capped poly(A)-tailed Renilla luciferase RNA havinga cap 0 structure was as follows if the RNA was capped using:N¹-methyl-GTP, 56%; O⁶-methyl-GTP, 167%; 2′-dGTP, 118%; 3′-dGTP, 80%;2′-OMe-GTP, 132%; 3′-OMe-GTP, 82%; 2′-F-2′-dGTP (i.e., 2′-F-dGTP), 55%;2′-azido-2′-dGTP (i.e., 2′-azido-dGTP), 99%; and 2′-amino-2′-dGTP (i.e.,2′-amino-dGTP), 148%.

The average relative translation efficiency ofmodified-nucleotide-capped poly(A)-tailed Renilla luciferase RNA havinga cap I structure in rabbit reticulocyte lysate was as follows if theRNA was capped using: N¹-methyl-GTP, 50%; O⁶-methyl-GTP, 122%; 2′-dGTP,84%; 3′-dGTP, 64%; 2′-OMe-GTP, 97%; 3′-OMe-GTP, 84%; 2′-F-2′-dGTP (i.e.,2′-F-dGTP), 107%; 2′-azido-2′-dGTP (i.e., 2′-azido-dGTP), 79%; and2′-amino-2′-dGTP (i.e., 2′-amino-dGTP), 144%.

In subsequent experiments in which a different luciferase transcript wasprepared from a firefly luciferase gene, and the transcript wassubsequently capped and polyadenylated using the same methods as for theabove modified-nucleotide-capped poly(A)-tailed Renilla luciferase RNA,the average relative translation efficiency of thismodified-nucleotide-capped poly(A)-tailed luciferase RNA having a cap Istructure in rabbit reticulocyte lysate was as follows if the RNA wascapped using: N¹-methyl-GTP, 16%; O⁶-methyl-GTP, 97%; 2′-dGTP, 128%;3′-dGTP, 98%; 2′-OMe-GTP, 170%; 3′-OMe-GTP, 137%; 2′-F-2′-dGTP (i.e.,2′-F-dGTP), 133%; 2′-azido-2′-dGTP (i.e., 2′-azido-dGTP), 107%; and2′-amino-2′-dGTP (i.e., 2′-amino-dGTP), 100%.

5B. In Vitro Translation in Wheat Germ Lysate.

By comparison with the GTP-capped poly(A)-tailed Renilla luciferase RNAhaving a cap 0 structure (100% translation efficiency), the relativetranslation efficiency in wheat germ lysate ofmodified-nucleotide-capped poly(A)-tailed Renilla luciferase RNA havinga cap 0 structure was as follows if the RNA was capped using:N¹-methyl-GTP, 31%; O⁶-methyl-GTP, 80%; 2′-dGTP, 54%; 2′-OMe-GTP, 36%;3′-OMe-GTP, 39%; 2′-F-2′-dGTP (i.e., 2′-F-dGTP), 53%; 2′-azido-2′-dGTP(i.e., 2′-azido-dGTP), 41%; and 2′-amino-2′-dGTP (i.e., 2′-amino-dGTP),90%.

The relative translation efficiency of modified-nucleotide-cappedpoly(A)-tailed Renilla luciferase RNA having a cap I structure in wheatgerm lysate was as follows if the RNA was capped using: N¹-methyl-GTP,31%; O⁶-methyl-GTP, 59%; 2′-dGTP, 54%; 3′-dGTP, 31%; 2′-OMe-dGTP, 47%;3′-OMe-GTP, 43%; 2′-F-2′-dGTP (i.e., 2′-F-dGTP), 61%; 2′-azido-2′-dGTP(i.e., 2′-azido-dGTP), 42%; and 2′-amino-2′-dGTP (i.e., 2′-amino-dGTP),110%.

1.-22. (canceled)
 23. A kit for obtaining a modified nucleotide-capped RNA comprising: (a) a capping enzyme system comprising an RNA guanyltransferase; and (b) a modified cap nucleotide, wherein the modified cap nucleotide: i) is 3′-deoxyguanosine-5′-triphosphate or comprises a modified 2′- or 3′-deoxyguanosine-5′-triphosphate, wherein the 2′- or 3′-deoxy position of the sugar moiety is substituted by a group other than a hydroxyl group or a hydrogen, in particular wherein the 2′- or 3′-deoxy position of the sugar moiety is substituted with an amino, an azido, a fluorine, a methoxy, a thiol, or a mercapto group, or wherein the O⁶-oxygen of the guanine base is replaced with a thiol or mercapto group; ii) comprises a modified guanosine-5′-triphosphate, wherein the 2′- or 3′-hydroxyl group of the ribose is substituted with an alkyl group, or wherein the O⁶-oxygen of the guanine is substituted with an alkyl group or is replaced with a thiol or mercapto group; or iii) is selected from: N¹-methyl-GTP; O⁶-methyl-GTP; 6-thio-GTP; 2′-O-methyl-GTP; 3′-O-methyl-GTP; 2′-amino-2′-dGTP; 3′-amino-3′-dGTP; 2′-azido-2′-dGTP; 3′-azido-3′-dGTP; 2′-F-2′-dGTP; 3′-F-3′-dGTP; 3′-dGTP; 2′-amino-2′,3′-ddGTP; 3′-amino-2′,3′-ddGTP; 2′-azido-2′,3′-ddGTP; and 3′-azido-2′,3′-ddGTP.
 24. The kit of claim 23, wherein the RNA guanyltransferase comprises a motif I that exhibits the amino acid sequence KTDG(I/V)x, wherein the sixth amino acid of said motif I is P, G, F, S or L.
 25. The kit of claim 23, further comprising uncapped RNA that exhibits a 5′ diphosphate.
 26. The kit of claim 23, further comprising polypeptide having RNA triphosphatase activity.
 27. The kit of claim 26, further comprising uncapped RNA that exhibits a 5′ triphosphate.
 28. The kit of claim 27, wherein the uncapped RNA is: (i) a primary RNA transcript from a human or animal patient, from a human, animal, plant, or fungal organism, organ, tissue, or cell, or from extracellular fluid from a patient or organism with a condition; (ii) primary RNA from an in vitro transcription reaction; (iii) obtained following fractionation of RNA from a biological source by subtractive hybridization and digestion, and RNA amplification that yields sense RNA; (iv) primary RNA from a prokaryotic source; (v) derived from a condition comprising a tumor or cancer cell; or (vi) derived from a pathogen, or from a eukaryotic cell that is infected by a bacterial, viral or fungal pathogen.
 29. The kit of claim 23, further comprising S-adenosyl-methionine or S-adenosyl-ethionine.
 30. The kit of claim 29, further comprising polypeptide having guanine-7-methyltransferase activity
 31. The kit of claim 30, further comprising modified-nucleotide capped RNA, wherein the 5′ cap nucleotide comprises a modified guanine nucleoside-5′-triphosphate of claim 23 (b).
 32. The kit of claim 31, further comprising an enzyme having mRNA (nucleoside-2′-O—) methyltransferase activity.
 33. The kit of claim 32, wherein the enzyme having mRNA (nucleoside-2′-O—) methyltransferase activity is: (i) encoded by a poxvirus DNA; (ii) encoded a vaccinia virus DNA; (iii) is purified from virions; (iv) is purified from a recombinant source; (v) is purified from E. coli cells that express a poxvirus gene that is cloned in a plasmid or other vector; (vi) is purified from E. coli cells that express the vaccinia gene that is cloned in a plasmid or other vector; (vii) is encoded by a yeast or fungal gene; or is encoded by a nematode, mammalian or other metazoan gene, whether from a wild-type or recombinant source.
 34. The kit of claim 31, wherein the modified-nucleotide capped RNA exhibits a cap with a cap1 structure, wherein the 5′ penultimate nucleotide has a 2′-O-methyl group.
 35. The kit of claim 23 further comprising poly(A) polymerase.
 36. The kit of claim 35, wherein said poly(A) polymerase is (i) Escherichia coli poly(A) polymerase; (ii) yeast poly(A) polymerase; or (iii) is recombinant poly(A) polymerase encoded by the E. coli pcnB gene.
 37. The kit of claim 31, wherein the modified-nucleotide capped RNA further exhibits a poly(A) tail.
 38. The kit of claim 34, wherein the modified-nucleotide capped RNA that exhibits a cap with a cap1 structure further exhibits a poly(A) tail.
 39. The kit of claim 23, further comprising a component that improves the efficiency of capping uncapped RNA that forms a duplex involving at least the 5′-terminal nucleotide, wherein said component consists of at least one of the following: (i) dimethylglycine (betaine); (ii) a single-stranded binding protein; (iii) an RNA helicase; (iv) an RNA polymerase; or (v) a DNA polymerase.
 40. The kit of claim 31, wherein the modified-nucleotide capped RNA further comprises a biotin moiety.
 41. The kit of claim 34, wherein the modified-nucleotide capped RNA that exhibits a cap with a cap1 structure further comprises a biotin moiety.
 42. The kit of claim 37, wherein the modified-nucleotide capped RNA that exhibits a poly(A) tail further comprises a biotin moiety. 